Hybrid molecules and their use for optically detecting changes in cellular microenvironments

ABSTRACT

The invention relates to methods and compositions which utilize the emission of light to monitor changes in microenvironments involving cells. The invention is especially useful for monitoring exocytotic activity such as detecting quantal release of synaptic vesicles. Fusion proteins of Cypridina luciferase and synaptotagmin-I or VAMP/synaptobrevin-2 were targeted to synaptic vesicles and, upon exocytosis, formed light-emitting complexes with luciferin present in the extracellular medium. Photon emissions in the presence of a depolarizing stimulus can be observed with these systems. pH-sensitive mutants of green fluorescent protein are also provided, which are useful for visualizing exocytosis and for imaging and measuring the pH of intracellular compartments.

This application claims priority from U.S. provisional application Nos.60/038,179 filed Feb. 13, 1997 and 60/036,805 filed Feb. 14, 1997 bothof which are incorporated herein by reference in their entirety.

TECHNICAL FIELD OF THE INVENTION

This invention relates to compositions and methods for monitoringchanges in the local environment inside or outside of cells by detectingoptical changes in an environment-sensitive reporter molecule. Inparticular, this invention relates to compositions which may bespecifically incorporated into cells, or may be caused to be produced bythem, which produce an optically detectable signal in response to achange in the environment in which the optically sensitive reportermolecule is present. This invention is particularly applicable todetecting the release of substances stored in exocytotic vesicles ofcells, such as synaptic vesicles, wherein the compositions of theinvention are localized in the vesicles and become exposed to theextracellular space upon release of the contents of the vesicles.

BACKGROUND OF THE INVENTION

Various methods are known for monitoring molecular changes in localenvironments at a microscopic level. Detection of changes in themicroenvironments involving active cells presents a particular challengebecause many methods involve destruction or damage to the cells beinganalyzed. Although significant information has been obtained usingelectrophysiological techniques, these techniques have limitedapplication for studying complex real time cell physiology and also maydamage and thereby alter the cells being recorded. Also,electrophysiology is limited to detecting events involving a change inelectrical potential.

Many cell process involve changes in the molecular microenvironment.Such processes include, for example, exocytosis and endocytosis whichinvolves contact between the intracellular and extracellularenvironments, and changes in ion concentration associated withelectrically active cells such as neurons and muscle cells. The releaseof cellular substances, in particular, is a generalized phenomenon ofseveral cell types which is fundamental to the function of multicellularorganisms. Blood cells, endocrine cells and neurons are examples of celltypes particularly dependent on such processes. Blood granulocytes, forexample, release various mediators of inflammation through exocytosis;endocrine cells release hormones required by other cells; and neuronsrelease neurotransmitters packaged in synaptic vesicles. The ability todetect changes in the microenvironment caused, for example, by thefusion of an exocytotic vesicle with a plasma membrane and contact ofthe lumenal surface and contents of the vesicle with the extracellularspace would provide information useful for understanding and modulatingprocesses which causes release of such cellular substances.

Exocytotic events are particularly important to the proper function ofneurons since synaptic transmission is dependent upon the controlledrelease of synaptic vesicles. Many problems in neurophysiology can bereduced to questions about the location, timing, and magnitude ofsynaptic activity, including, for instance, the integration of inputs bya single neuron, synaptic plasticity, and pattern classification andstorage by neural networks. The study of these and related problemswould greatly benefit from a method that allows direct recording frommany synapses simultaneously, with the capacity to reliably detectsingle exocytotic events. Such a method would appear optimal because, onthe one hand, central synapses generally transmit information via thefusion of a single synaptic vesicle (1,2), while, on the other hand, thecomputational power of the nervous system arises from networkscontaining large numbers of synapses (refs. 3-5).

While current electrophysiological methods allow the activity ofindividual synapses to be recorded, they do not permit populations to bestudied. There is a practical limit to the number of cells that can beimpaled simultaneously with intracellular electrodes, and importantly,an invasive method requires an a priori decision on which cells tostudy, making discovery difficult. Extracellular field recordings withmultiple electrodes avoids some of these problems and thereby allows thecollective activities of many cells to be measured, but does not permitactivity to be ascribed to individual synapses or neurons (6,7). Opticalimaging of light emission from fluorescent indicators of membranepotential or intracellular Ca²⁺ concentration (7-10) greatly increasesspatial resolution but again, does not measure synaptic activitydirectly. An alternative optical approach that offers a direct gauge ofsynaptic activity is to load synaptic vesicles with fluorescent dyes andto observe dye release (11,12). However, this method is intrinsicallyincapable of resolving individual quanta, which can cause only a smalldecrease in total fluorescence. Despite their limitations, thesetechniques have opened a window on multicellular phenomena as diverse asthe representation of visual scenes by retinal ganglion cells (13) andthe emergence of cortical circuits during development (14). Methods thatreveal the detailed patterns of synaptic inputs and outputs in entirenetworks can thus be expected to disclose important new physiologicalconcepts operative at the relatively unexplored interface betweencellular and systems neurophysiology.

Due to the importance to physiology of proper exocytotic processes inneurons and other cells types, it is therefore desirable to developsensitive compositions and methods which can detect changes in themicroenvironment inside or outside of cells including quantal exocytoticevents in real time. Molecules which can detect changes inmicroenvironments would be useful as probes of cellular events involvingchanges in such microenvironments due to movement of molecules insolution or the spacial location of molecules associated with cellmembranes. It would be particularly desirable to have availablemolecules that provided an optical signal upon encountering such achange in the microenvironment.

Various types of molecules have been used in the art for the detectionof the presence of other molecular entities. Radiochemical labels havehigh sensitivity but are hazardous and must be used with appropriatecaution. In addition, these labels are not useful for real timelocalization. Optical labels such as fluorescent molecules or otherforms of dyes have also been coupled to molecules to act as reportersfor the detection of specific molecular entities. Typically a reportercable of generating an optical signal is bound to a specific bindingmolecule which is a member of a ligand binding pair. Such bindingmolecules are usually antibodies, specific binding proteins, e.g.receptors or peptide hormones which specifically binds a correspondingtarget ligand. These reporter-ligands are reagents which must be addedfrom the external environment to the system under investigation. Theirusefulness is therefore limited by their accessibility to theappropriate molecular target, nonspecific binding or diffusion toinappropriate locations and availability of appropriate binding pairs.Another type of molecular reporter comprises signal generating moleculesexpressed endogenously by a cell. Several bioluminescent proteins havebeen reported as useful as detectable labels for optically reporting thepresence of a molecular entity.

The green fluorescent protein (GFP) of Aequora victoria, for example, isa naturally fluorescent protein with a p-hydroxybenzylideneimidazolonechromophore, created by in vivo cyclization and oxidation of thesequence Ser-Tyr-Gly (positions 65-67). The chromophore's phenolicgroup, derived from Tyr-66, exists in two states of protonation, whichin all likelihood underlie the protein's two main excitation peaks at395 and 475 nm (ref. 47). Several reports have characterized variousbioluminescent proteins. See, for example, Cormier et al., “RecombinantDNA Vectors Capable of Expressing Apoaequorin”, U.S. Pat. No. 5,422,266;Prasher, “Modified Apoaequorin Having Increased BioluminescentActivity”, U.S. Pat. No. 5,541,309; Cormier et al., “Isolated RenillaLuciferase And Method Of Use Thereof”, U.S. Pat. No. 5,418,155; McElroyet al., “Recombinant Expression of Coleoptera Luciferase”, U.S. Pat. No.5,583,024. The use of bioluminescent fusion proteins as reporters ofgene expression has also been reported. See, for example, Harpold etal., “Assay Methods And Compositions For Detecting And Evaluating TheIntracellular Transduction Of An Extracellular Signal”, U.S. Pat. No.5,436,128; Tsein et al., “Modified Green Fluorescent Proteins”.International Application WO 96/23810; Gustafson et al., “FusionReporter Gene For Bacterial Luciferase”, U.S. Pat. No. 5,196,524; andChalfie et al. “Uses Of Green-Fluorescent Protein”, U.S. Pat. No.5,491,084. Although GFP has been reported as a useful reporter molecule,its utility would be further enhanced if it could be made sensitive tochanges in the microenvironment.

SUMMARY OF THE INVENTION

This invention provides compositions and methods useful for detectingchanges in microenvironments. Many dynamic systems are dependent oncompartmentalization of molecules with subsequent merging ofcompartments or release of the contents of one compartment into anothercompartment. Endocytosis and exocytosis are examples of biologic systemswhich involve compartmentalization of molecular entities. Thecompositions and methods of this invention are especially useful fordetecting changes in the microenvironment associated with changes incompartmentalization. Detection of quantalcellular exocytotic events inreal time such as those occurring in connection with the release ofsynaptic vesicles is made possible by this invention.

The method of this invention comprises detecting a change in the lightemitting properties of a hybrid molecular reporter present in a firstcompartment upon contact with a second compartment. The hybrid molecularreporter comprises a targeting region and a reporter region whichparticipates in a light-generating reaction upon contact with the secondcompartment. As applied to cells, the method of this invention enablesthe quantal detection of the release of components of exocytoticvesicles, especially synaptic vesicles. The method of this invention isanticipated to be applicable to detecting the release of vesicularcontents within a cell as well.

This invention also provides hybrid molecular reporter molecules. Atleast two types of hybrid molecules are provided by this invention. Onetype of molecule is derived externally from the compartment in which itis to be introduced. When used to detect cellular processes, such hybridmolecules are typically not genetically encoded by the cells to whichthey are directed. In addition, the light-generating component providesa detectable optical signal which is environment sensitive. The othertype of hybrid molecule provided by this invention is geneticallyencoded. These molecules themselves are of two types: a) those whichinclude a targeting region and a reporter region wherein the reporterregion is co-expressed with the targeting region and is capable ofgenerating an optically detectable signal; and b) those which include atargeting region and a binding region which binds to a separate reportermolecule capable of generating an optically detectable signal but whichis not encoded by the cell encoding the targeting-binding region andwhich is provided from the external environment.

The hybrid molecules of this invention are typically, but notnecessarily, polypeptides since polypeptides are particularly wellsuited as targeting entities and may be genetically encoded andexpressed. The compositions of this invention may be targeted to varioustypes of multicompartment systems. In one embodiment, the hybridmolecules may be targeted to liposomes and used to monitor delivery tocells of substances, such as drugs contained by the liposomes. Inanother embodiment, the compositions of this invention may be targetedto intracellular locations, such as exocytotic vesicles, where they arenot in contact with the extracellular environment until an exocytoticevent occurs. Upon exocytosis, the interior of the exocytotic vesicle,including for example the lumenal side of the vesicle membrane, comes incontact with the extracellular environment. By coming into contact withthe extracellular environment the compositions of this inventiontargeted to the vesicle cause a release of photons which may be detectedas an optical event indicative of quantal exocytotic release.

The targeting polypeptide of the invention preferably is targeted toexocytotic vesicle membranes and the amino acid sequence required forgeneration of the optical signal is preferably located at the lumenalsurface of the vesicle membranes. This embodiment encompasses moleculeswhich generate an optical signal that directly reports neurotransmitterrelease. The detection of individual vesicle fusion events is madepossible by this invention which also provides for the regeneration ofprobes for many rounds of recording. Preferred probes for this and otherembodiments are genetically encoded proteins.

Genetic control of the expression of the probes of this invention allowsrecordings to be obtained from cells, cultures, tissue slices or exposedtissues of transgenic animals, and affords means to detect individualcells including neurons (by localized DNA transfer techniques), types ofneurons (by cell-type specific promoters), or elements of a circuit (byrecombinant viral vectors that spread through synaptic contacts). Suchprobes are useful for detecting the release of synaptic vesicle.

In one embodiment of this invention, hybrid reporter molecules whichcomprise a luciferase enzyme and at least a portion of a vesiclemembrane protein are provided. These hybrid reporter molecules arereferred to as “synaptolucins”.

This invention also includes mutants of green fluorescent protein ofAequora victoria which exhibit environment sensitive excitation and/oremission spectra and are useful, for example, as reporter moieties inthe hybrid molecules of this invention. Examples ofenvironment-sensitive GFP mutants provided by this invention are variouspH sensitive mutants, which are termed “pHluorins”. Two preferred typesof GFP mutants which are provided by this invention are mutants which,in response to a reduction in pH, from pH 7.4 to 5.5 exhibit attenuationor loss of the GFP excitation peak at 475 nm (ecliptic pHluorins) andmutants which exhibit an inverse in the ratio of the excitation peaks at395 and 475 nm upon a reduction in pH from 7.4 to 6.0 (ratiometricpHluorins). The nucleic acid molecules encoding the amino acid sequencesof these GFP mutants are also within the scope of this invention.

In another embodiment of this invention, hybrid reporter molecules whichcomprise a pHluorin and at least a portion of a vesicle membrane proteinare provided. These hybrid reporter molecules are termed“synaptopHluorins”.

In another embodiment of this invention, nucleic acid molecules areprovided which encode for the hybrid reporter molecule polypeptides ofthis invention. Preferably, such nucleic acid molecules comprise apromoter which causes expression of the polypeptide in a specific cell.Also provided are vectors and transformed cells containing the nucleicacid molecules of this invention. Various types of cells may betransformed with the nucleic acids of this invention and include primarycells either in vivo or in vitro, cultured cells including cell linesand cells of transgenic animals. Transgenic animals which express theclaimed nucleic acids are thus another embodiment of this invention.

The hybrid molecules and methods of this invention are useful fordetecting contact of molecules in an environment with a secondenvironment. This invention is particularly useful for detecting fusionevents associated with cell membranes involving endo or exocytosis. Inaddition, fusion of liposomes containing the hybrid reporter moleculeswith cells may also be monitored according to this invention. Screeningfor molecules which alter exocytotic processes, especially in specificcell populations is also made possible by this invention. In addition,the method of this invention provides a means of simultaneouslyrecording the activity of several cells, for example neuronal cells,because one can distinguish spatially, the source of multiple opticalsignals generated by the cellular release of the peptides provided bythis invention.

By providing a means of detecting release of exocytotic vesicles indiscrete cell populations this invention provides a means of identifyingthe contribution of specific proteins or cell processes to exocytosis bymeasuring such processes in cells which have been altered in some way,for example by the inactivation of certain genes believed to encode forcertain proteins involved in such exocytotic processes. Thus, forexample, the use of this invention with animals or cells in whichcertain genes have been “knocked out” may provide useful models forproviding information regarding exocytosis in various cell types andunder various conditions.

Through the use of the synaptopHluorins of this invention, synaptictransmission at individual boutons, as well as secretion in a variety ofcell types may be non-invasively imaged by fluorescence microscopy. Itis anticipated that the use of pHluorins can be extended to visualizesuch diverse trafficking processes as endocytosis, receptor activation,and intercompartmental translocation in individual cells or populationsof designated cell types, in cultures, tissues, or intact transparentorganisms.

The GFP mutants provided by this invention are useful as optical labels.These mutants may be bound to a specific binding molecule which is onemember of a ligand binding pair to detect the presence of the othermember of the ligand binding pair. These mutants may also be used asreporters of protein expression. Because of their pH sensitivity, thesemutants may also be used to detect pH changes in their environment.

An object of this invention is to provide bifunctional polypeptideswhich may be localized to specific cell types, cell compartments, orcell locations and which participate in the generation of an opticalsignal upon contact of the polypeptide with the extracellular space.

Another object of this invention is to provide a method for opticallydetecting the release of the contents of exocytotic vesicles, especiallyfor example, synaptic vesicles.

Another object of this invention is to provide a method of identifyingprocesses and substances which regulate vesicle release.

Yet another object of this invention is to provide mutants of GFP whichexhibit excitation and/or emission spectra which are sensitive tochanges in the environment.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1. Cofractionation of synaptolucins with synaptic vesicle proteins.Amplicon-infected PC12 cells were homogenized and postnuclearsupernatants sedimented into 5-25% glycerol gradients. Small synapticvesicles band in fractions 4-9, endosomes in fractions 11-14 (ref. 25).The bottom fractions contain material collected on sucrose cushions.Compared to the slower-sedimenting fractions, only 15% of this materialwas analyzed by SDS-PAGE. Proteins were precipitated withtrichloroacetic acid, separated on 8-18% gels, and transferred tonitrocellulose. The filters were probed with mAb M48 (ref. 23), directedagainst synaptotagmin-I (top), or mAb CL67.1 (ref. 24), directed againstVAMP-2 (bottom). Bound antibodies were visualized by ECL (Amersham;Arlington Heights, Ill.).

FIGS. 2A-2F. Hippocampal neurons expressing synaptolucin-1, imaged bywide-field microscopy. About 60% of the neurons were infected by HSVtransducing synaptolucin-1. Scale bar, 20 μm. FIG. 2A displays thesynaptic map, revealed by loading nerve terminals with FM 4-64. Thefluorescent signal from FM 4-64 was acquired at low intensifier gain andaveraged over 32 video frames. FIGS. 2B-2E represent photonregistrations accumulated from synaptolucin emissions over 30 seconds,obtained at 30 nM luciferin, maximum image intensifier gain, and adiscriminator value for photon detection that suppressed background andequipment noise (photon counts in the presence of a depolarizingstimulus but the absence of luciferin) to an average of 1.06 photonregistrations per 100-pixel field. The preparation was imagedsuccessively in normokalemic solution (2B) and during three hyperkalemicchallenges to induce exocytosis, performed in either the presence (FIGS.2C and 2D) or the absence (FIG. 2E) of external Ca²⁺. Ten minutes underresting conditions elapsed between each of the successive stimuli. Thedashed red lines in FIGS. 2A-2C mark areas of stimulation-independentsynaptolucin activity, in all likelihood due to virus-infected glialcells. Panel 2F superimposes the synaptolucin signal of FIG. 2C, coloredhere in red, onto a binary version of the synaptic map. The binary mapwas constructed by thresholding FIG. 2A such that pixels with anintensity above the 97^(th) percentile appear in black.

FIG. 3. Matched filtering of two synaptolucin images, FIGS. 2B (control)and 2C (exocytosis triggered), with their common synaptic map, FIG. 2A,as shown schematically in FIG. 2F. The x- and y-axes indicate therelative shifts between filter and image in these projections, and theordinate the normalized cross-correlation function, a measure of thematch between image and filter (Ref. 38). The function is computed bypointwise multiplication in the spatial frequency domain and—due to theproperties of the Fourier transform—periodic (Ref. 38). Only a singleperiod, from −π to π in the x- and y-directions, is shown. At shift(0,0), filter and image are in register, at shifts (x,+/−π) or (+/−π,y),the filter's center is displaced to an edge of the image. (Top) Scanningof FIG. 2C, showing evoked synaptolucin emissions. Note the peak at afilter shift of (0,0), indicating a matching structure in the image.(Bottom) Scanning of FIG. 2B, lacking evoked synaptolucin emissions.Note the absence of a central peak.

FIG. 4. Hippocampal neurons expressing synaptolucin-1, imaged bywide-field microscopy at the same intensifier and detector settings asin FIG. 2. (Top) Photon registrations from synaptolucin emissions duringthe first 30 sec after triggering exocytosis, colored in red andsuperimposed on synaptic maps obtained with FM 4-64. (Bottom) FM 4-64images after a 30-sec hyperkalemic challenge. Panels 4A and 4B wererecorded before and panels 4C and 4D after treatment with BoNTs B and F.BoNTs were applied during a 5-min depolarization (to enhance toxinuptake into recycling synaptic vesicles) and then during a 3-hincubation in complete medium with 1 μM tetrodotoxin. Note the markeddecrease in FM 4-64 fluorescence intensity in panel 4B as opposed topanel 4D. Scale bar, 20 μm.

FIG. 5. Amino acid sequence of wild-type GFP (SEQ ID NO: 1).

FIG. 6. cDNA nucleic acid coding sequence of wild-type GFP (SEQ IDNO:2).

FIG. 7A. cDNA nucleic acid coding sequence of GFP mutant 1B11t (SEQ IDNO:3).

FIG. 7B. cDNA nucleic acid coding sequence of GFP mutant 14E12t (SEQ IDNO:4).

FIGS. 8A-8D. Excitation spectra of GFP mutants. The excitation spectrafrom 360 to 500 nm was determined at an emission wavelength of 510 nm atpH 7.4 (solid lines) and pH 5.5 (dashed lines) for wild type GFP (FIG.8A), GFP mutant S202H (FIG. 8B), GFP mutant 1B11t (FIG. 8C) and GFPmutant 14E12t (FIG. 8D).

FIGS. 9A-9F. Excitation spectra of 1B11-like GFP mutants. Excitationspectra were obtained as described for FIGS. 8A-8D (clone 1D10, FIG. 9A;clone 2F10, FIG. 9B; clone 2H2, FIG. 9C; clone 1B11, FIG. 9D; clone 8F6,FIG. 9E; and clone 19E10, FIG. 9F).

FIGS. 10A-10G. Excitation spectra of 14E12-like GFP mutants. Excitationspectra were obtained as described for FIGS. 8A-8D (clone 14E12, FIG.10A; clone 14C9, FIG. 10B; clone 14C8, FIG. 10C; clone 2G3, FIG. 10D;clone S202H, FIG. 10E; clone 14D9, FIG. 10F; and clone 8H8, FIG. 10G).

FIG. 11A. cDNA nucleic acid sequence for the coding region of clone 1D10(SEQ ID NO:5).

FIG. 11B. cDNA nucleic acid sequence for the coding region of clone 2F10(SEQ ID NO:6).

FIG. 11C. cDNA nucleic acid sequence for the coding region of clone 2H2(SEQ ID NO:7).

FIG. 11D. cDNA nucleic acid sequence for the coding region of clone 1B11(SEQ ID NO:8).

FIG. 11E. cDNA nucleic acid sequence for the coding region of clone 8F6(SEQ ID NO:9).

FIG. 11F. cDNA nucleic acid sequence for the coding region of clone19E10 (SEQ ID NO:10).

FIG. 12A. cDNA nucleic acid sequence for the coding region of clone14E12: 14E12 (SEQ ID NO:11).

FIG. 12B. cDNA nucleic acid sequence for the coding region of clone 14C9(SEQ ID NO: 12).

FIG. 12C. cDNA nucleic acid sequence for the coding region of clone 14C8(SEQ ID NO: 13).

FIG. 12D. cDNA nucleic acid sequence for the coding region of clone 2G3(SEQ ID NO: 14).

FIG. 12E. cDNA nucleic acid sequence for the coding region of cloneS202H (SEQ ID NO: 15).

FIG. 12F. cDNA nucleic acid sequence for the coding region of clone 14D9(SEQ ID NO:16).

FIG. 12G. cDNA nucleic acid sequence for the coding region of clone 8H8(SEQ ID NO:17).

FIG. 13. cDNA nucleic acid sequence for the coding region of Cypridinaluciferase (SEQ ID NO: 18)

FIGS. 14A-14B. Side (14A) and top (14B) views of the beta-barrelstructure of GFP. The polypeptide backbone is shown in grey and thechromophore, formed by internal cyclization of the tripeptideSer⁶⁵-Tyr⁶⁶-Gly⁶⁷, in green; the hydroxyl group of Ty⁶⁶ is highlightedin red. In FIG. 14A, blue regions indicate cassettes of amino acids(positions 94-97, 146-149, 164-168, 202-205, 221-225) subjected tocombinatorial mutagenesis. In FIG. 14B, blue regions indicate the 7 keyresidues and their side chains. The predominant side chain conformationof Thr²⁰³ is shown.

FIG. 14C. Excitation spectra at 508 nm of wild-type GFP.

FIG. 14D. Excitation spectra at 508 nm of ratiometric pHluorin (cloneC6).

FIG. 14E. Excitation spectra at 508 nm of ecliptic pHluorin (clone 8F3).

The ordinate scales in FIGS. 14C-14E reflect normalized differences inemitted fluorescence intensity, recorded at 25° C. Samples contained27.5 μM chromophore, 50 mM sodium cacodylate, 50 mM sodium acetate, 100mM NaCl, 1 mM CaCl₂, and 1 mM MgCl₂.

FIG. 15A. The relationship between R_(410/470) (mean±SD) and pH. Cellsexpressing GPI-anchored ratiometric pHluorin at their surface (n=28)were imaged in imaging buffers adjusted to pH values between 5.28 and7.8 (at 37° C.).

FIG. 15B. Ratiometric pH measurement of extracellular space, colorencoded according to the look-up table displayed at right. The targetingmodule used was a GPI-anchor for delivery to the cell surface oftransiently transfected HeLa cells, in imaging buffer of pH 7.4).

FIG. 15C. Ratiometric pH measurement of endosomes, color encodedaccording to the look-up table displayed at right. The targeting moduleused was Cellubrevin, in transiently transfected HeLa cells. Scale bar,10 μm, is valid for FIGS. 15B-15E.

FIG. 15D. Ratiometric pH measurement of the trans-Golgi network, colorencoded according to the look-up table displayed at right. The targetingmodule used was TGN38, in transiently transfected HeLa cells.

FIG. 15E. Ratiometric pH measurement of synaptic vesicles, color encodedaccording to the look-up table displayed at right. The targeting moduleused was VAMP/synaptobrevin, in hippocampal neurons infected with an HSVamplicon vector.

FIG. 16A. Map of all synapses in a field of hippocampal neurons,obtained by immunostaining with a monoclonal antibody againstsynaptotagmin-I. Note the numerous synaptic inputs to the cell body onthe lower right.

FIG. 16B. Map of synaptopHluorin-expressing synapses, formed byHSV-infected neurons whose somata lie outside the field of view. Due tothe low multiplicity of infection, only a small fraction of the synapseslabeled in FIG. 16A are synaptopHluorin-positive. Note the relativepaucity of synaptopHluorin-positive inputs to the cell body on the lowerright, attesting to the specificity of synaptopHluorin expression.Arrows indicate points of registration between FIGS. 16A and 16B. Scalebar, 20 μm.

FIG. 16C. Photo: the dashed box in FIG. 16B, shown at highermagnification. Two boutons (1 and 2) are identified.

FIG. 16D. Recordings of synaptic activity obtained from the two boutonsidentified in FIG. 16C. R_(410/470) was sampled at 1 Hz, with 200-msecexposures at each excitation wavelength. Neurons were stepped throughtwo depolarization cycles by raising extracellular [K⁺], first in 2 mMextracellular Ca²⁺ (abscissa 0-40 sec) and then in the absence of Ca²⁺and in the presence of 2.5 mM EGTA (abscissa 90-130 sec).

FIG. 17A. Secretion visualized with ecliptic pHluorin. RBL-2H3 cellsexpressing ecliptic pHluorin were pre-incubated for 3 h with 10 ng/mlanti-DNP IgE to load cell surface IgE receptors. Under restingconditions (frame 1), secretory granules are visible only with 410-nmbut not with 470-nm excitation. After addition of 1 mg/ml anti-IgEantibodies to trigger a secretory response, 200-msec exposures wereacquired at 0.1 Hz. Selected frames (2-7) taken with 470-nm excitationare displayed. All images were contrast-enhanced and low-pass filteredwith a 3×3 binomial kernel. Scale bar, 10 μm.

Frame 4: arrows mark a cluster from which individual spots aredisappearing in frames 5 and 6.

Frames 5-7: a “flickering” spot.

FIG. 17B. The relationship between optical and biochemical measures ofsecretion. Times at which individual frames in FIG. 17A were acquiredare indicated by arrows. The activity of β-hexosaminidase, a markerenzyme of RBL cell granules, was assayed in 20-μl aliquots of imagingbuffer, withdrawn at the indicated times.

FIG. 17C. Size distribution of secretory events, derived from raw imagesacquired with 470-nm excitation. Integrated fluorescence intensity isthe product of pixel area and average intensity. Events were countedif: 1) the grey level of each of their pixels exceeded a threshold whichwas empirically set so that no events would be scored in the absence ofa secretory stimulus (frame 1) and

2) their area exceeded 4 contiguous pixels, equal in size to thein-focus image (Airy disk) of a fluorescent object with a diameter ofca. 0.5 μm, somewhat smaller than the average mast cell granule.

FIG. 18. DNA sequence of coding region of GFP mutant C6 (SEQ ID NO:19)

FIG. 19. Amino acid sequence of GFP mutant C6 (SEQ ID NO:20).

FIG. 20. DNA sequence of coding region of GFP mutant 8F3 (SEQ ID NO:21).

FIG. 21. Amino acid sequence of GFP mutant 8F3 (SEQ ID NO:22).

FIG. 22. Amino acid sequence of GFP 1B11t (SEQ ID NO:23).

FIG. 23. Amino acid sequence of GFP 14E12t (SEQ ID NO:24).

FIG. 24. Amino acid sequence of GFP 1D10 (SEQ ID NO:25).

FIG. 25. Amino acid sequence of GFP 2F10 (SEQ ID NO:26).

FIG. 26. Amino acid sequence of GFP 2H2 (SEQ ID NO:27).

FIG. 27. Amino acid sequence of GFP 1B11 (SEQ ID NO:28).

FIG. 28. Amino acid sequence of GFP 8F6 (SEQ ID NO:29).

FIG. 29. Amino acid sequence of GFP 19E10 (SEQ ID NO:30).

FIG. 30. Amino acid sequence of GFP 14E12 (SEQ ID NO:31).

FIG. 31. Amino acid sequence of GFP 14C9 (SEQ ID NO:32).

FIG. 32. Amino acid sequence of GFP 14C8 (SEQ ID NO:33).

FIG. 33. Amino acid sequence of GFP 2G3 (SEQ ID NO:34).

FIG. 34. Amino acid sequence of GFP S202H (SEQ ID NO:35).

FIG. 35. Amino acid sequence of GFP 8H8 (SEQ ID NO:36).

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to compositions and methods for detecting changesin microenvironments useful for monitoring dynamic physiologicalprocesses.

Hybrid Polypeptide Compositions

The compositions of this invention include hybrid molecules comprising atargeting region and a reporter region capable of participating in areaction resulting in an optically detectable signal when the hybridmolecule encounters a change in the microenvironment. These hybridmolecules are preferably polypeptides comprising at least one amino acidsequence which targets the hybrid molecule to a specific cell orintracellular location and at least one other amino acid sequence whichfunctions as the reporter and participates in generating the opticallydetectable signal. Modifications of the basic structure are within thescope of this invention including, for example, molecules comprising aplurality of reporter amino acid sequences as a means of amplifying theoptical signal. Whereas in many cases it will be desirable to use anamino acid sequence as the reporter region, other moieties capable ofgenerating an optical signal such as fluorescent or fluorogenicmolecules may also be used as the reporter region. As used herein, afluorogenic molecule is a molecule which takes part, as a reagent orcatalyst, in a bioluminescent or chemiluminescent reaction, or whichtakes part in a reaction which generates a fluorescent or luminescentspecies. Fluorogenic substrates are molecules whose processing by anappropriate enzyme results in the emission of light, for exampleluciferin. A linker comprising at least one amino acid may also beinterposed between the targeting and reporter regions.

Another form of hybrid molecule according to this invention aremolecules comprising a targeting region and a specific binding regionwhich functions as one member of a ligand-binding pair which isrecognizable by a separate molecular entity which functions as thereporter. Such hybrid molecules would therefore bind to affinity-basedreagents which include, but are not limited to, antibodies, or fragmentsthereof, and other members of ligand-binding pairs. Preferably, thehybrid molecule is a polypeptide which is genetically encodable. Thesehybrid molecules may be endogenous or engineered structures. Theseparate light-generating reporter, such as a labeled antibody, whichbinds to the specific binding region of the hybrid molecule is generallynot expressed by the cells expressing the hybrid molecule but is addedto the system from the external environment.

The ability to detect the change in the microenvironment results fromthe combination of the choice of targeting region and reporter region.For example, a reporter region which when expressed would beconstitutively capable of generating an optical signal may be turned offby being expressed as part of a molecule comprising a targeting regionwhich targets the molecule to a region of the cell having an environmentwhich does not provide the necessary environment to generate thedetectable signal. Upon a change in the microenvironment, the reporterregion would then generate the detectable optical signal. This change inmicroenvironment may result, for example, due to movement of the hybridmolecule to a different location resulting in exposure to theextracellular space where necessary substrates may be present or achange in pH occurs.

The hybrid polypeptide compositions of this invention are particularlyuseful for monitoring physiological changes in the microenvironment suchas those which occur during exocytotic processes when the exocytoticvesicle fuses with the plasma membrane causing the internal contents ofthe vesicle to come in contact with the extracellular environment. Tomonitor such processes it is possible through this invention to loadintracellular vesicles with the hybrid polypeptides of this inventionand then detect the fusion of the vesicle with the plasma membrane. In aparticularly preferred embodiment neuronal synaptic vesicles are causedto contain the hybrid molecules of this invention to enable the quantaldetection of synaptic vesicle release.

Incorporation of the hybrid molecules of this invention in liposomesalso provides a method for detecting the contact of the liposome andfusion with cell membranes. Liposomes are increasing being used as drugdelivery systems. By inserting the hybrid reporter molecules of thisinvention into either the interior of the liposome or into its lipidbilayer one may then detect fusion of the liposome with a targetmembrane. One method of practicing this embodiment for example would beto load vesicles with a hybrid reporter molecule where the reportermoiety is self-quenching fluorophore. Upon fusion of the liposome withthe target cell the fluorophore would be diluted causing an increase influorescence.

Nucleic Acid Molecules

Another embodiment of this invention are the nucleic acid moleculesencoding the hybrid polypeptides. These nucleic acid molecules compriseat least one nucleic acid sequence encoding the targeting amino acidsequence in reading frame with at least one nucleic acid sequenceencoding the reporter amino acid sequence. An additional nucleic acidsequence encoding an optional flexible amino acid linker may also beinterposed in between the targeting and reporter encoding nucleic acidsequences. The length of the amino acid linker may be chosen to optimizeaccessibility of the reporter region of the molecule to the externalenvironment while maintaining sufficient anchorage with the targetregion to maintain the molecule in the desired location. In oneexemplary embodiment the linker is fifteen (15) or fewer amino acids inlength. In another embodiment the linker is twelve (12) amino acids inlength. Preferred amino acid linkers are —(Ser-Gly-Gly)₄ and—(Ser-Gly-Gly)₂-Thr-Gly-Gly.

The nucleic acid molecule of the invention preferably comprises apromoter sequence which causes expression of the hybrid polypeptide in adesired tissue and/or at a desired time. Thus, the promoter sequencealso can contribute to targeting of the hybrid polypeptide. Tissuespecific promoters are described in Short, “Nucleic Acid ConstructEncoding A Nuclear Transport Peptide Operatively Linked To An InduciblePromoter”, U.S. Pat. No. 5,589,392 which is incorporated herein byreference in its entirety, but see in particular Col. 8-10. Examples ofpreferred promoters include the promoters for the genes encodingsynaptotagmin-I and VAMP/synaptobrevin-2 when the target is neuronalsynaptic vesicles. Promoters for polypeptide hormones such as insulin(Bucchini et al., Proc. Natl. Acad. Sci., USA, 82:7815-7819 (1985),growth hormone, prolactin, (Crenshaw et al., Genes and Development3:959-972 (1989), and proopiomelanacortin (Tremblay et al., Proc. Natl.Acad. Sci., USA, 85:8890-8894 (1988) which are released by exocytosismay be useful for targeting the hybrid molecules of the inventionrespectively to pancreatic beta cells, and various cells of thepituitary gland, depending on the genes they express.

Neuron-specific promoters may be used for targeting the hybrid moleculesof this invention to the nervous system and specific neurons. Suchpromoters generally fall within two categories, those which are“neuron-specific housekeeping” promoters, and those which are cell-typespecific. Neuronal “house-keeping” promoters, for example, may beselected from the promoter for the synapsin I gene (See, Sauerwald A. etal., J. Biol. Chem. 265:14932-14937 (1990) and neuron specific enolase(Sakimura K. et al., Gene 60:103-113 (1987). Promoters for specific celltypes may include the tryptophan hydroxylase promoter for serotoninergicneurons (Stoll J. and Goldman, D., J. Neurosci. Res. 28:457-465 (1991);the choline acetyltransferase promoter L. B. J. Neurochem. 61:306-314(1993); the dopamine beta hydroxylase promoter for adrenergic neurons(Shaskus, J. et al., J. Biol. Chem. 267:18821-18830 (1992).

The nucleic acid molecules of this invention may be inserted in variousvectors, preferably viral vectors such HSV or adenovirus, to causeexpression in the desired cells. The constructs for use in thisinvention should also contain the necessary initiation, termination, andcontrol sequences for proper transcription and processing of the geneencoding the hybrid polypeptides of this invention. Target cells for thenucleic acid molecules of this invention may be in vitro or in vivo.

Methods for inserting DNA into cells are well known in the art and havealso been reported in connection with introducing bioluminescentproteins into cells. For example, the following patents and patentapplication, which are all incorporated herein in their entirety byreference, refer to methods for using bioluminescent polypeptides asreporters of gene expression. Tsien et al., “Modified Green FluorescentProteins” International Application WO 96/23810; Gustafson et al.,“Fusion Reporter Gene For Bacterial Luciferase”, U.S. Pat. No.5,196,524; and Chalfie et al. “Uses Of Green-Fluorescent Protein”, U.S.Pat. No. 5,491,084. Methods for introducing the nucleic acids into cellsinclude for example conventional gene transfection methods such ascalcium phosphate co-precipitation, liposomal transfection (see Epand etal. U.S. Pat. No. 5,283,185 (which is incorporated herein in itsentirety by reference), microinjection, electroporation, and infectionor viral transduction. In addition, it is envisioned that the inventioncan encompass all or a portion of a viral sequence—containing vector,such as those described in P. Roy-Burman et al., U.S. Pat. No. 5,112,767(which is incorporated herein by reference in its entirety) fortargeting delivery of genes to specific tissues.

Transgenic animals, preferably non-human mammals such as mice or rats,or genetically tractable organisms such as C. elegans, Drosophila, andzebrafish, may also be produced which express the hybrid polypeptidesencoded by the nucleic acid molecules of this invention. Methods forpreparing transgenic animals are described in the art in Hogan et al.,Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor,N.Y. (1987); Short, “Nucleic Acid Construct Encoding A Nuclear TransportPeptide Operatively Linked To An Inducible Promoter”, U.S. Pat. No.5,589,392; and Wagner et al., “Virus-Resistant Transgenic Mice”, U.S.Pat. No. 5,175,385 (all of which are incorporated herein by reference intheir entirety).

Targeting Amino Acid Sequences

The amino acid sequences which target the hybrid molecules of thisinvention may be entire proteins or fragments thereof which cause thehybrid molecule to be substantially localized to the desired targettissue and/or cell region. Transmembrane proteins specific for celltypes are preferred for use with this invention and may include forexample, receptors, ion channels, cell adhesion proteins (e.g. N-CAM) orvesicle docking proteins (e.g. synaptotagmin) and transport proteins.Antibodies, Fc fragments or other specific binding proteins may also beused since they would cause the hybrid molecule to be bound to specificantigenic recognition sites.

Preferably the polypeptides used for targeting the hybrid molecules aretargeted to sites that are subject to changes in their microenvironment.For example, if the targeting moiety causes the hybrid molecule to belocated intracellularly but becomes exposed to the extracellular spacethe hybrid molecule can be used to detect the time and location of suchexposure to the extracellular environment provided the extracellularenvironment has the properties or substrate necessary to activate thereporter region of the hybrid molecule. In addition, it is preferablethat such targeting polypeptides not be freely diffusible so as to avoiddilution of the optical signal and to keep the optical signalconcentrated and localized at its site of generation. Accordingly,membrane proteins are especially preferred for use as the targetingregion of the hybrid molecules of the invention. For detecting synapticvesicle release synaptotagmin, VAMP/synaptobrevin are preferred.

In a preferred embodiment of this invention the hybrid molecules areengineered proteins consisting of two modules: a targeting modulederived from a synaptic vesicle-specific integral membrane protein, anda light-generating module that is constitutively “off” but becomesactivated when the vesicle fuses with the presynaptic membrane. In thisway, light emission signals synaptic activity. Potential targetingmodules may include any amino sequence which comprises at least oneregion which is lumenally exposed to provide an attachment site for theluminescent module. The targeting modules of this invention comprise anamino acid sequence which upon expression provides for targeting thesequence to a specific cellular location. In this case of synapticvesicles this would preferably be the lumenal surface of synapticvesicles. Such amino acid sequences preferably include a sequencesufficiently homologous to an endogenous protein to cause the sequenceto be transported to the desired location. The targeting module maycomprise a plurality of regions including at least one targeting regionand at least one luminescent attachment site. The luminescent attachmentsite is not restricted to any particular portion of the targeting moduleprovided it is accessible to the luminescent module if other portions ofthe targeting module are membrane bound or embedded within a membrane.

Reporter Regions

The reporter regions of the hybrid molecules of this invention may beany molecular moiety that participates in a bioluminescent,chemiluminescent, fluorescent, or fluorogenic reaction, or whichparticipates in the quenching or suppressing of fluorescence orluminescence. Changes in light intensity or wavelength may be used tooptically detect the presence or activation of the reporter moiety. Suchreporter activity may result from ionic changes such as those resultingin pH changes, quenching, presence or absence of enzyme substrates, orability to bind a second reagent, such as an antibody conjugate, whichitself participates in generating an optically detectable signal.

Light-generating reporters typically are two-component systems, wherelight emitted by one component undergoes either a spectral or anintensity change due to a physical interaction of the first componentwith the second component. According to this invention, the firstcomponent may either be co-expressed as a fusion protein with thetargeting region of the hybrid molecules, or it may bind to a specificbinding site attached to the targeting molecule (see below). The secondcomponent of the light-generating system may be a molecule or ion, theconcentration of which differs or can be made to differ in the variouscompartments which the first component contacts. An optical signal,and/or a change in the optical signal, is generated by movement of thelight-generating component between the different compartments, or bycontact of the light-generating component with different compartments.

Non-limiting examples of suitable light-emitting reporters, referred toherein as “fluorogens”, for use with this invention include:

1. enzyme-substrate complexes (e.g. luciferases)

2. environmentally sensitive fluorophores as the first component, whichcan be genetically encodable (e.g., an environment-sensitive GFP mutant,infra) or not genetically encodable such as synthetic dyes such as, forexample, fluorescein, SNAFL, SNARF, NERF, merocyanines, fura-2, etc. Thesecond component which activates these light-generating componentsincludes, but is not limited to, ions (e.g. protons, calcium ions, andany other endogenous or synthetic ions); or any other molecule whichconcentration differs, or can be made to differ, between the differentcompartments to be analyzed;

3. fluorescence resonance energy transfer pairs; and

4. self-quenching fluorophores, which exhibit increased fluorescenceupon dilution into a larger compartment, such as the extracellularspace.

The non-genetically encoded light-generating reporters may be bound tothe member of the ligand pair which recognizes and binds the othermember of the ligand binding pair associated with the target region inthe compartment to be analyzed.

Where the hybrid molecule of this invention is itself to be added tocells for localization followed by activation by a reporter notexpressed by the cells to be analyzed, the reporter may be any form ofsubstance discussed above which is covalently bound to the targetingamino acid sequence such as an antibody or antibody fragment.Preferably, however, the reporter itself is an amino acid sequence whichis co-expressed as a fusion protein with the targeting amino acidsequence as discussed above. Under such circumstances the optical signalis generated by a reporter amino acid sequence which is selected fromthe group consisting of a) amino acid sequences which contain apH-sensitive chromophore or fluorophore, b) self-quenching fluorescentamino acid sequences which fluoresce upon dilution into a largercompartment (for example, into the extracellular space), c) enzymaticsequences which react with a fluorogenic substrate present in theextracellular space, and d) amino acid sequences which bind to aspecific immunological reagent present in the extracellular space.

Several bioluminescent molecules are known in the art and are suitablefor use with this invention as the reporter of the change inmicroenvironment. For example Cormier et al., “Recombinant DNA VectorsCapable of Expressing Apoaequorin”, U.S. Pat. No. 5,422,266, which isincorporated herein in its entirety by reference, refers to recombinantDNA vectors capable of expressing the bioluminescent proteinapoaequorin. In the presence of coelenterate luciferin, apoaequorin istermed aequorin and produces visible light in the presence of calciumions. Various natural and modified luciferases are known. See, forexample, Prasher, “Modified Apoaequorin Having Increased BioluminescentActivity”, U.S. Pat. No. 5,541,309, Tsien et al., supra. Examples ofother luciferases suitable for use with this invention include Cormieret al., “Isolated Renilla Luciferase And Method Of Use Thereof”, U.S.Pat. No. 5,418,155; McElroy et al., “Recombinant Expression ofColeoptera Luciferase”, U.S. Pat. No. 5,583,024. Harpold et al., “AssayMethods And Compositions For Detecting And Evaluating The IntracellularTransduction Of An Extracellular Signal”, U.S. Pat. No. 5,436,128 andthat obtained from Cypridina hilgendorfii (29).

Green-fluorescent protein (GFP) is described in Chalfie et al. “Uses OfGreen-Fluorescent Protein”, U.S. Pat. No. 5,491,084. Certain modifiedforms of green-fluorescent protein have been reported and may be usedwith this invention, for example as described in Tsien et al., “ModifiedGreen Fluorescent Proteins” International Application WO 96/23810. TheGFP mutants described herein are particularly preferred for use asreporters in this invention.

For measurement of synaptic release the emitted signal must be ofsufficient intensity to be detected upon release of a single synapticvesicle. In addition, photon emission must be conditional upon synapticvesicle exocytosis. In one embodiment, a luciferase acting on amembrane-impermeant substrate may be employed. If the substrate ispresent in the extracellular medium and the enzyme sequestered insynaptic vesicles, light emission can not occur, but once the vesiclefuses with the presynaptic membrane and the catalytic module isexternalized, a burst of photon emission follows (FIG. 2C). Preferably,a useful luciferase is one that can i) catalyze a sufficiently highphoton flux for imaging (a quantity dependent on the eniyme's turnovernumber and the quantum yield of the light-emitting complex), ii) use amembrane-impermeant substrate, iii) be capable of folding in the ERlumen and be targeted to synaptic vesicles, and iv) operate efficientlyunder the pH and salt conditions of the extracellular environment.

Of the well-characterized bioluminescent systems, that of the ostracodCypridina (or Vargula) hilgendorfii (29) matches this profile remarkablywell and is preferred. The commonly used firefly luciferase is lesspreferred for the reasons discussed below. Cypridina luciferase is amonomeric, naturally secreted glycoprotein of 62 kDa (16,30) which canbe expressed in and is secreted from transfected mammalian cells (31),whereas firefly luciferase is peroxisomal. Cypridina luciferin (32)carries a guanidino group expected to be positively charged atphysiological pH and to thereby render the molecule slowly permeant oreven impermeant to membranes. Unlike firefly luciferase, which requiresATP (and, for sustained activity, coenzyme A), Cypridina luciferase usesno cofactors other than water and O₂ (29). Its luminescent reactionproceeds optimally at pH 7.2 and physiological salt concentrations (30),whereas that of firefly is optimal at low ionic strength (activity isinhibited 5- to 10-fold by physiological salt), alkaline pH, andreducing conditions. With a turnover number of 1600 min⁻¹ (33) and aquantum yield of 0.29 (34), Cypridina luciferase produces a specificphoton flux exceeding that of the optimized firefly system (35) by afactor of at least 50.

The GFP mutants provided by this invention may also be used as reporterssince they too may be expressed as fusion proteins with the targetregions.

Another form of reporter for use with this invention includes a bindingregion which is bound to the targeting region, which binding regionspecifically binds a separate light-generating reporter molecule. Thebinding region is preferably genetically encodable, and is mostpreferably co-expressed as a fusion protein with the targeting aminoacid sequence. The separate light-generating reporter molecule to whichthe binding region specifically binds may be an antibody, a fragment ofan antibody such as a Fab fragment, or any other moiety which is aligand partner of the binding region attached to the targeting region.The separate light-generating reporter molecule will be labeled with acomponent which participates in producing a fluorogenic signal may thenbe detected by an appropriate method.

Any member of a ligand binding pair is suitable for use as the bindingregion provided the other member may be covalently attached to, orotherwise stably associated with, a light-generating reporter molecule.Preferably the binding regions are amino acid sequences recognizable byspecific antibodies. Examples of such amino acid sequences which arerecognized by commercially available antibodies include, but are notlimited to:

1) myc-tag: EQKLISEEDL

antibody: 9E10

Evan, G. I., et al., (1985) Mol. Cell. Biol. 4:2843-2850,

2) Flag-tag: DYKDDDDK

antibody: M2

Brizzard, B. L., et al., (1994) BioTechniques 16: 730-734,

3 VSV-tag: YfDIEMNRLGK

antibody: P5D4

Kreis, T. E. (1986) EMBO J. 5:931-941, and

4) HA-tag: YPYDVPDYA

antibody: 12CAS

Wilson, I. A., et al., (1984) Cell 37: 767-778.

By expressing different binding regions in a single transgenic animal itis possible according to this invention to identify differentpopulations of cells in a single preparation since differentlight-generating reporters may then be used.

Detection of the optical signal generated by the reporter may beaccomplished using standard methods known in the art. Images may berecorded with a video or digital camera attached to a standard orfluorescent microscope, digitized and optically enhanced. Methods foramplifying the optical signal such as through the use of an intensifierphotocathode may also be employed. Control images obtained prior toactivation of the reporter region may also be digitized, electronicallystored and subtracted from the image to remove background emissions tomore accurately identify specific activation events such as thoseresulting from exocytosis.

As shown in the examples herein, the methods and compositions of thisinvention are especially useful for detecting release of exocytosis, asin synaptic vesicle release. The reproducible pattern of photonregistrations in repeated trials (compare FIGS. 2C and 2D) attests tothe reliability of synaptolucin and synaptopHluorin hybrid molecules ofthis invention as indicators of exocytosis, with many possibleapplications. For example, the interpretation of many studies onsynaptic plasticity is fraught with controversy, possibly because thepre- and postsynaptic components of neurotransmission cannot bedistinguished by traditional methods, which are all indirect (40).Measuring exocytosis directly via synaptolucins or synaptopHluorins,before and after maneuvers that alter synaptic strength, can help toresolve ambiguities. Or, the multiple inputs to a postsynaptic neuroncan be mapped using synaptolucins or synaptopHluorins. If thepostsynaptic neuron's membrane potential is monitored electrically, theshape of a synaptic potential as it arrives after propagation throughthe dendritic tree could be measured and immediately correlated with itsanatomical site of origin, with important implications for inputintegration (5). The pattern of activation of individual synaptic inputscan be measured in relation to the activation of a post-synaptic neuron,affording a direct means of establishing firing rules.

The sensitivity and temporal resolution afforded by synaptolucins andsynaptopHluorins are determined by three factors: the number ofsynaptolucin or synaptopHluorin molecules per vesicle, their specificemission rate, and the time over which photon counts can be integratedto keep pace with the relevant physiology. At video rates, this intervalis usually a single video frame, or about 30 msec. At the other extreme,with long photon-counting times as in the present experiments, thetiming of the synaptic vesicle cycle itself becomes limiting. In suchcases, photon emission begins as a vesicle fuses with the presynapticmembrane—probabilistically (41,42), at any time during the observationperiod—and ends as the vesicle's reporter molecules arere-internalized—again, probabilistically—or when the camera shutter isclosed. Vesicle recycling will terminate synaptolucin activity virtuallyinstantaneously: if luciferin is taken up by recycling vesicles at itsbulk concentration of 30 nM, only about one in a thousand recycledvesicles will contain a luciferin molecule (as can be calculated fromthe internal diameter of a synaptic vesicle of 50 nm), and those fewwhich do will consume the internalized luciferin (via luciferase)rapidly. This effectively prevents the visualization of endocytosedvesicles, and limits photon emissions to the synaptolucin dwell time inthe presynaptic membrane. In the case of the synaptopHluorins, theduration of fluorescence upon vesicle re-internalization will depend onthe rate at which the intra-vesicular pH is re-established. It isanticipated that the synaptopHluorins will be useful for studying thisphenomenon.

When using a Cypridina luciferase, luciferin should be used atsubsaturating concentrations to minimize its penetration into cells,limiting luciferase to about 3% of its maximum velocity. Use of a trulyimpermeant luciferin derivative at saturating concentrations wouldincrease photon emissions about 35-fold. In addition, the number oflight-generating modules per synaptic vesicle can be increased i) bygene replacement technology to fully substitute synaptolucins for VAMPand/or synaptotagmin and ii) by including multiple light-generatingmodules in a single synaptolucin. Singly, or in combination, theseengineering steps should permit reliable detection of single vesiclefusion events at every visible synapse.

The compositions and methods provided by this invention are useful fordetecting changes in microenvironments, particularly those involvingcells wherein different cell compartments contact each other or come incontact with the extracellular space. This invention is especiallyuseful for detecting exocytotic events, especially the release ofsynaptic vesicles.

The ability to monitor such events is useful for providing new means ofdiagnosing disorders involving alterations of exocytosis. In addition,model systems involving cell cultures may be used to determine theeffects of drugs on exocytotic processes. Not only may effects onrelease be monitored by this invention, but the rate of re-uptake ofreleased material may also be monitored by detecting the rate ofdecrease in the signal intensity over time as dilution of the hybridmolecules occurs. This invention may therefore be particularly usefulfor identifying new drugs such as antidepressants which alter theneuronal release and/or re-uptake of neurotransmitters.

The pHluorins of this invention are useful for imaging many traffickingprocesses that connect compartments of differing pH. Secretory storagevesicles generally have an acidic pH (50), so that pHluorins, even inthe form of a single VAMP-based construct (see FIGS. 16 and 17), can beused to monitor exocytosis. The controlled release of the contents ofsecretory vesicles underlies a great many intercellular signallingprocesses in homeostasis and development. Imaging of such events incells, or in genetically tagged cell types in tissues or transgenicorganisms, is of value in cell and developmental biology, as well as inphysiology. The pHluorin method can also be adapted for high-throughput,cell-based screening for compounds affecting trafficking processes ofmedical relevance. Examples of transport pathways connecting the cellsurface and acidic endosomes (50) include receptor-mediated endocytosis,the internalization of activated signalling receptors (such as tyrosinekinases (75) and G protein-coupled receptors (76)), and thetranslocation of glucose transporters to the cell surface in response toinsulin (77).

SynaptopHluorins are anticipated to find many neurobiologicalapplications. Because the probes are encodeable, specific types ofneurons (for example, glutamatergic vs. GABAergic) can be tagged andrecorded selectively. Because synaptopHluorins rely exclusively oncellular machinery for synthesis and localization, as well as fortranslating neurotransmission into optical signals, the activities ofentire populations of neurons can be imaged in situ. This combination ofself-sufficiency with anatomical and functional specificity promises toaid in visualizing the flow of information in complex neural systems.

As discussed above, this invention may be used with cell lines orprimary cultures. In addition tissue slices from animals including thetransgenic animals of this invention may be used. This invention mayalso be used on dissociated cells, organ cultures and dissected andexposed tissues of intact animals which are bathed in an appropriatemedium. Because of its relative ease of accessibility, neurons of themyenteric plexus of intact animals are particularly well suited foranalysis using the compositions and methods of this invention.

Where the hybrid molecules are added to the cells from the medium thecells are incubated with the medium for a sufficient time, typicallyseveral hours or overnight, to allow the molecules to be taken up by thecells and packaged for release. After an appropriate time the mediumcontaining the hybrid molecule, is washed out and replaced with mediumcontaining the necessary substrate or condition such as pH to causeactivation of the reporter upon its contact with the extracellularspace. Cells which have been made to express the hybrid molecules of theinvention may be contacted with medium containing the activatingconditions directly.

Environment-sensitive GFP Mutants (pHluorins)

Mutants of GFP of Aequora victoria are provided according to thisinvention by mutating amino acids which directly or indirectly interactwith the natural chromophore p-hydroxybenzlideneimidazole, which iscreated by the in vivo cyclization and oxidation of the GFP Ser-Tyr-Glysequence (positions 65-67).

Wild-type GFP has a bimodal excitation spectrum (54) with peaks at 395and 475 nm (FIG. 1C). Underlying the two excitation maxima areprotonated and deprotonated states of the chromophore (55-57), which arestabilized by different conformations of a hydrogen bond networkinvolving the phenolic oxygen of Tyr⁶⁶, which is incorporated into thechromophore (55, 48, 58). The protonated form of Tyr⁶⁶ accounts for the395-nm excitation maximum, while deprotonated Tyr⁶⁶, present in asmaller fraction of GFP, gives rise to the minor 475-nm peak (55-57).Excitation at either 395 or 475 nm results in virtually identicalemission spectra, with a single maximum around 508 nm.

The excitation spectrum of GFP is essentially unaltered between pH 5.5and 10 (54). Because water can access critical residues like Tyr⁶⁶, asevidenced by a pronounced deuterium kinetic isotope effect (56) onfluorescence excited at 395 nm, this lack of pH sensitivity implies thatprotonation-deprotonation reactions are conformationally constrained. Inother words, a given GFP is kinetically trapped in either of twoalternate conformations, in one of which the chromophore is protonated(and can be excited at 395 nm), and in the other of which it isdeprotonated (and can be excited at 475 nm).

Wild-type GFP as obtained commercially (Clontech, Inc.) has the aminoacid sequence depicted in FIG. 5. This sequence differs from thatpublished in patent application WO 96/23810 in that a Thr is present,rather than an Ile, at position 161. Mutants of GFP may be identified bydetermining the excitation spectrum over a range of about 350 to about500 nm at an emission wavelength of 508 nm. Alterations in the naturallyoccurring excitation peaks at 395 and 475 nm may thus be determined.Similarly, alterations in the emission spectrum may also be determinedby scanning the emission spectrum from about 350 to about 500 nm at aconstant excitation wavelength, preferably 395 nm which is the peakexcitation wavelength at pH 7.4.

Amino acid residues in contact with, or in close proximity to, the GFPchromophore identify preferred regions for substituting one or moreamino acids. Amino acids identified as being in preferred regionsinclude Gln-69, Gln-94, Arg-96, Asn-121, His-148, Phe-165, Ile-167,Gln-183, Thr-203, Ser-205 and Glu-222. Most preferred are Gln-94,Arg-96, His-148, Ile-167, Thr-203, Ser-205, and Glu-222.

Amino acid substitutions are preferably made at residues flanking theabove-identified amino acids since side chains of the flanking residueswhich are present in the beta-barrel structure face the outer surface ofthe protein and may therefore be more sensitive to the environment,especially-changes in pH. To create a pH sensitive mutant having asensitivity at a particular pH range it is preferred to substitute oneor more of the flanking amino acids identified above with amino acidshaving a side chain with a pKa within the range for which the mutant GFPis to be used to detect pH changes. Preferably the range is about one pHunit above and below the pKa. For example, histidine, having a pKa valueof about 6.4, is preferred for obtaining mutants having pH sensitivityfrom about pH 5.5 to 7.5; glutamic acid, with a pKa of about 4.3, ispreferred for mutants having a pH sensitivity of about 3.3 to 5.3; andlysine, having a pKa of 10.8, is preferred for mutants having asensitivity of between about 9.8 to 11.8. Other substitutions may bemade based on the known pKa's for particular amino acids.

Among the various positions which may be suitable for substitution witha pH sensitive amino acid, position 202 is preferred. Additional randommutagenesis of the regions in contact with or, in close proximity to,the chromophore may also be introduced to further modify the lightemitting properties of the mutant GFP protein. Such additional mutationsmay further alter the light-emitting properties of GFP by quenchingemitted photons or altering excitation properties of the protein.

The terminology used herein for referring to amino acid substitutions inthe GFP mutants employs the notation XNY, where “X” is the single letteror three-letter amino acid code for the amino acid residue at position“N” in wild-type GFP, and “Y” is the single letter or three-letter codefor the amino acid inserted at position N in place of X.

Amino acid substitutions suitable for preparing the GFP mutants of thisinvention include, but are not limited to, S147D, S147E, S147P, N149H,N149V, N149Q, N149T, N149L, N149D, N149Y, N149W, T161I, K163A, K166Q,I167V, R168H, S175G, and S202H. Preferred are S147D, N149Q, N149D,T161I, V163A, K166Q, I167V and S202H. More preferred combinations arethose represented by clones 1B11t, 14E12t, 8F3, and C6. Most preferredare clones 8F3 and C6. The V163A and S175G mutations may optionally beincluded to decrease the temperature sensitivity of the mutant GFPs.

GFP mutants prepared according to the methods of this invention may alsocontain substitutions of amino acids of the wild type protein with aminoacids of similar charge. See Yang et al., “The Molecular Structure OfGreen Fluorescent Protein,” Nature Biotechnology, 14:1246-1251 (1996)which is incorporated herein by reference for a report of the shape andtopology of GFP.

Two classes of pH sensitive mutants are provided by way of example. Themutants of one class exhibit attenuation or loss of the excitation peakat 475 nm and a loss of fluorescence intensity excitable at 395 nm.Preferred members of the class include the clones 8F3, 1D10, 2F10, 2H2,1B11, 8F6 and 19E10. See Table 2, infra. Most preferred is clone 8F3.The second class of pH sensitive mutants responds to decreases in pHwith decreased fluorescence due to decreased excitation at the 395 nmpeak and increased fluorescence due to increased excitation at the 475nm peak. Preferred members of this class include clones C6, 14E12, 14C9,14C8, 2G3, S202, H14D9, and 8H8. Most preferred is C6.

Mutants of the 8F3 class (ecliptic pHluorins) are preferred for use fordetecting release of secretory granules. Secretory granules contain aproton pump of the vacuolar type. The proton pump generates aproton-motive force (pmf) across the synaptic vesicle membrane,consisting of a pH gradient (vesicle lumen acidic, pH ca. 5.5) and amembrane potential (vesicle lumen positive). When a synaptopHluorinutilizing 8F3 as the light-generating module is targeted to a secretoryvesicle, the acidic intravesicular pH will turn the excitation peak at475 nm off. Light emission after excitation at 475 nm, however, willresume at the more alkaline pH prevalent in the synaptic cleft, allowingan optical signal to be associated with exocytosis. This signal is largeenough to permit detection of individual vesicle fusion events in realtime.

The dynamic range of the signal generated by mutant C6 between pH 7.4and 5.5 is too small to allow detection of individual vesicle fusionevents. However, C6 and other ratiometric pHluorins, fused to anappropriate targeting module, are ideally suited to serve as ratiometricpH indicators for dynamic compartments such as intracellular organelles.

The GFP mutants and the Cypridina luciferase provided by this inventionmay be used for any application for which it is desirable to have alight-emitting detectable signal. Such mutants may therefore be used todetect the presence of an analyte in standard immunometric assays bycoupling the GFP mutant or the Cypridina luciferase to an appropriatebinding ligand using techniques well known in the art for conjugatingproteins to other molecules. The GFP mutants and the Cypridinaluciferase may also be expressed as fusion proteins and act as reportersof expression. Bound to such fusion proteins, the environment sensitivemutants may be used to spatially detect in real time the location of theexpressed protein.

The nucleic acid molecules encoding the GFP mutants and the Cypridinaluciferase are also within the scope of this invention. The nucleic acidsequences encoding the amino acid sequences disclosed in Table 2, aswell as the specific nucleic acid sequences of FIG. 5 (GFP) and FIG. 13(Cypridina luciferase), are preferred. These nucleic acid molecules maybe incorporated into plasmids, and expression vectors for expression invarious cells including bacteria cells such as E. coli, fungi, such asyeast, and mammalian cells by methods well known in the art. Expressionin nearly transparent animals such as C. elegans and zebra fish ispreferred.

Alterations to the nucleic acid sequences disclosed herein, which do notalter the amino acid sequence of the encoded polypeptide but which maketranslation of the nucleic acid sequence more efficient with respect tothe preferred codon usage of the expressing cells, are within theability of those skilled in the art, and nucleic acid sequences modifiedin this manner are also contemplated to be within the scope of thisinvention.

EXAMPLES Example 1 Synaptolucins

Synaptolucins. Total Cypridina RNA extracted with TRISOLV (Biotecx;Houston, Tex.) served as the template for the RT-PCR synthesis of a cDNAencoding the luciferase (16). The PCR product was subcloned into theamplicon plasmid pα4“a” (17,18) and its sequence determined withSEQUENASE 2.0 (United States Biochemical; Cleveland, Ohio). To constructsynaptolucins-1 and -2, the appropriate portions of the open readingframes for Cypridina luciferase (16) and, respectively, ratsynaptotagmin-I (19) and VAMP/synaptobrevin-2 (20,21) were fused viastretches of nucleotides encoding the flexible linker —(Ser-Gly-Gly)₄—.

The amplicon plasmids were transfected into E5 cells (17,22) with thehelp of LIPOFECTAMINE (Gibco BRL; Bethesda, Md.), and replicated andpackaged into virions after infection with 0.1 PFU/cel of the HSVdeletion mutant d120 (22). The primary virus stock was passaged on E5cells until the vector-to-helper ratio exceeded 1:4; the ratio wasestimated as the number of synaptolucin-positive Vero cells (byimmunostaining, using mAbs M48 (23) and CL67.1 (24)) vs. the number ofviral plaques formed after infection of E5 cells.

PC12 Cells were infected at a multiplicity of 1 amplicon virion/cell andat 6 h p.i. harvested in buffer H (10 mM HEPES-NaOH, pH 7.4, 150 mMNaCl, 0.1 mM MgCl₂, 1 mM EGTA, 1 mM PMSF and 1 μg/ml each of aprotinin,leupeptin and pepstatin). Homogenates were prepared by 13 passes througha ball-bearing cell cracker and postnuclear supernatants (5 min at 5,000g) fractionated on 5-25% (w/v) glycerol gradients in a Beckman SW41rotor, operated for 2 h at 41,000 rpm (25). Gradient fractions wereanalyzed by SDS-PAGE, Western blotting, and immunostaining.

To measure synaptolucin activities, postnuclear supernatants weresolubilized on ice with 1% NONIDET P-40, clarified (10 min at 15,000 g),and diluted 50-fold into buffer L (20 mM HEPES-NaOH, pH 7.4, 150 mMNaCl, 2 35 mM CaCl₂, 2 mM MgCl₂) that had been prewarmed to 30° C. Afteraddition of Cypridina luciferin to 5 1 μM, photon fluxes were integratedfor 10 sec in an LXB 1250 luminometer calibrated with a [¹⁴C]hexadecanestandard (26). Synaptolucin concentrations were determined byquantitative immunoblotting with mAbs M48 (23) and CL67.1 (24) and[¹²⁵I]protein G (New England Nuclear; Boston, Mass.), using therecombinant cytoplasmic domains of synaptotagmin and VAMP as thestandards.

Hippocampal Neurons. The hippocampal CA1-CA3 fields of P1 Sprague-Dawleyrats were dissected into EBSS with 10 mM HEPES-NaOH, pH 7.0, andmechanically dissociated after treatment with 20 U/ml papain(Worthington; Freehold, N.J.) (27,28). Cells were plated onto thepoly-D-lysine- and laminin-coated surface of 35-mm dishes with central8-mm glass windows (adhesive substrates were from Sigma Chemical Corp.;St. Louis, Mo.). The cultures were maintained in BME with Earle's saltsand 25 mM HEPES-NaOH, pH 7.4, supplemented with 20 mM glucose, 1 mMsodium pyruvate, 10% fetal bovine serum, 0.1% MITO+Serum Extender(Collaborative Biomedical Products; Bedford, Mass.), 100 U/mlpenicillin, 0.1 mg/ml streptomycin, and from day 5 after plating, 5 μMcytosine arabinoside (Sigma Chemical Corp.) (28). The preparations wereinfected with HSV amplicon vectors after 1-2 weeks in vitro. Viralinocula were diluted to multiplicities of roughly 0.1 in conditionedmedium containing 1 mM kynurenate (Fluka; Buchs, Switzerland), adsorbedfor 1 h, removed, and replaced with conditioned medium.

Optical Recording. At 8-20 h p.i., culture dishes were transferred to aPDMI-2 microincubator (Medical Systems Corp., Greenvale, N.Y.) mountedon the stage of a Zeiss AXIOVERT 135 TV microscope and held at 30° C. Ateflon insert forming an 8 mm wide channel across the optical window wasplaced in the dish to allow rapid perfusion with either normokalemicsolution (25 mM HEPES-NaOH, pH 7.05, 119 mM NaCl, 2.5 mM KCl, 2 mMNaCl₂, 2 mM MgCl₂, 30 mM glucose) or its hyperkalemic counterpart (KClraised to 90 mM, NaCl reduced to 31.5 mM). Nerve terminals were stainedby a 1-min exposure to 3 μM FM 4-64 (Molecular Probes; Eugene, Oreg.) inhyperkalemic solution (12) with 1% dialyzed bovine serum, followed bysuperfusion with normokalemic solution for >10 min.

FM 4-64 fluorescence was excited with the 510-560 nm band of anattenuated xenon arc lamp; alternatively, synaptolucin bioluminescencewas initiated by adding 30 nM luciferin from a 30 μM methanolic stock.Emitted light was collected with a Zeiss 40×/1.3 NA PLAN-NEOFLUAR oilimmersion objective, 590 nm longpass-filtered in the case of FM 4-64fluorescence, and focussed onto the photocathode of a C2400-30H imageintensifier coupled to a C2400-75 charge-coupled device (both fromHamamatsu Photonics; Hamamatsu, Japan). The video signal was 8-bitdigitized in an ARGUS-20 image processor (Hamamatsu Photonics) and savedto a POWER MACINTOSH for analysis, using NIH Image 1.60(http://rsb.info.nih.gov/nih-image/), TRANSFORM 3.3 (Fortner ResearchLLC; Sterling, Va.), and MATHEMATICA 3.0 (Wolfram Research; Champaign,Ill.).

The cDNA encoding Cypridina luciferase was used to construct twosynaptolucins. In synaptolucin-1, the C-terminus of luciferase was fusedto the N-terminus of synaptotagmin-1, located in the lumen of synapticvesicles. The hybrid protein relies on the cleavable signal peptideencoded by the luciferase gene for membrane translocation and isanchored in the membrane of the synaptic vesicle by the transmembranedomain of synaptotagmin. In synaptolucin-2, the mature N-terminus ofluciferase is fused to the C-terminus of VAMP-2, located in the vesiclelumen. This results in a second type of hybrid protein with amembrane-anchor segment that also serves as a non-cleavable signalsequence. The luciferase cDNA which was obtained (GenBank accessionnumber U89490) differed from the published DNA sequence (ref. 16) at 30positions, only three of which gave rise to amino acid substitutions:Asp-16 →Val, Ile-346 →Leu, and Asn-495 →Ser. The first substitutionshifted the predicted signal peptide cleavage site (16), leading us toconsider Gln-19 the mature N-terminus and to construct synaptolucin-2accordingly. The cDNA nucleic acid sequence encoding the Cypridinaluciferase, which is also considered part of this invention, is shown inFIG. 13.

When expressed in PC12 cells, the synaptolucin genes directed thesynthesis of membrane proteins of the expected sizes which co-sedimentedwith their respective targeting modules in velocity gradients (FIG. 1).The synaptolucins, like VAMP and synaptotagmin, were found both insynaptic vesicles (fractions 4-9) and endosomes (fractions 11-14) (25).Both synaptolucins were enzymatically active, with a k_(cat) of 5.2 and3.7 photon emissions sec⁻¹ per synaptolucin-1 and -2 molecule,respectively.

Imaging Neurotransmitter Release. Initial experiments on hippocampalneurons, performed at a saturating luciferin concentration of 5 μM (30),revealed that a depolarizing stimulus was not required for photonemissions to occur. It was suspected that this signal arose from theintracellular synaptolucin pool, which would become visible if Cypridinaluciferin, an imidazo[1,2-a]pyrazine nucleus with mostly hydrophobicsubstitutions (32), crossed biological membranes once its guanidinogroup was deprotonated. Thus, the pH of the bath solutions was loweredfrom 7.4 to 7.05, to favor the protonated luciferin species, and theluciferin content was decreased to reduce diffusion across membranes.Indeed, at a luciferin concentration of 30 nM, the background signaldisappeared and photon emissions became stimulation-dependent. However,longer photon-counting times were required because at a luciferinconcentration so far below the K_(m) of luciferase (0.52 μM, ref. 30),synaptolucin operated at only 3% of its V_(max).

FIG. 2 illustrates a typical imaging experiment on hippocampal neuronsinfected with an HSV amplicon vector transducing synaptolucin-1. Wefirst obtained a map of the synapses within the field of view (FIG. 2A)by taking the preparation through a depolarization cycle (12) in thepresence of FM 4-64 (36), a member of the family of fluorescent dyesthat are known to stain recycling synaptic vesicles (11). FM 4-64 waschosen over the more widely used FM 1-43 (11,12) because it does notabsorb significantly at 462 nm, the emission wavelength of Cypridinaluciferin (34), and thus permits the acquisition of an unperturbedsynaptolucin signal from a stained preparation. After perfusion withnormokalemic solution for at least 10 min, sufficient to replenish thesynaptic vesicle pool and to remove excess FM 4-64, a bolus of luciferinwas added to the bath solution and photon emissions were counted for thenext 30 sec. In many cases, such as the one shown in FIG. 2B, somephotons were registered in the absence of a depolarizing stimulus, butthese originated mainly from regions without an appreciable density ofsynapses (compare the areas marked by dashed red lines in FIGS. 2A and2B). It is likely that HSV-infected glial cells in the mixed culture arethe source of this background signal, because the neurotropism of HSV inhippocampal cultures is incomplete (17), and because synaptotagmin, thetargeting module of synaptolucin-1, appears at the cell surface whenexpressed in non-neuronal cells (37). More precise targeting may beaccomplished by using neuron-specific promoters.

To record light emission resulting from synaptic activation, thehippocampal preparation was perfused with hyperkalemic solution todepolarize the neurons, open voltage-gated Ca²⁺ channels in presynapticterminals, and trigger exocytosis. Because Cypridina luciferin isunstable in aqueous solution, decomposing with a t_(½) on the order of 1min (29,30), a second bolus of luciferin was added immediately afterdepolarization, and photons were counted for 30 sec thereafter. A fargreater number of photons were registered than in the absence of astimulus, and now their pattern (FIG. 2C) was similar to the synapticmap recorded with FM 4-64 (FIG. 2A). The match, however, was imperfect,presumably because the synaptolucin image contained background emissionsfrom virus-infected glial cells (compare the areas marked by dashed redlines in FIG. 2B and 2C), and because only a subset of synapses (thoseformed by neurons that are virus-infected) are potential light sources.Repeating the depolarization after a 10-min resting period evoked asimilar but not entirely identical response (FIG. 2D), whereasdepolarization without Ca²⁺ influx (by omitting free Ca²⁺ from thehyperkalemic solution) left the photon count at baseline level, withphoton emissions only from the regions attributed to glial cells(compare FIG. 2E and 2B).

To examine the degree of correspondence between the sites ofsynaptolucin activity and the synaptic map more rigorously,photon-counting images were overlaid with a binary filter constructedfrom the synaptic map, FIG. 2A, as depicted schematically in FIG. 2F.The filter was chosen such that it “transmitted” only at pixels wherethe intensity of FM 4-64 fluorescence exceeded the 97^(th) percentile ofthe grayscale (black areas in FIG. 2F) but blocked transmissionelsewhere (gray areas in FIG. 2F). If such a digital filter scansanother image and the intensity of the transmitted signal is plotted asa function of the relative shift between filter and image, maxima occurwhere the filter detects a matching structure in the image (ref. 38).FIG. 3 shows the result of scanning two synaptolucin images, FIGS. 2Band 2C, with a filter constructed from FIG. 2A. Clearly, the signal inFIG. 2B has no counterpart in the synaptic map, supporting itsidentification as a contaminant of non-neuronal origin (FIG. 3, bottom).The sites of evoked photon emissions in FIG. 2C, by contrast, produce asharp maximum where filter and image are in register and thus map tonerve terminals (FIG. 3, top).

In addition to characteristic sensitivities to membrane potential andextracellular Ca²⁺, an optical signal generated by synaptic vesicleexocytosis should be susceptible to clostridial neurotoxins thatinactivate components of the machinery for transmitter release (39).FIG. 4 shows an experiment performed to address this point. An FM 4-64/synaptolucin image pair was first acquired to locatesynaptolucin-expressing synapses (FIG. 4A). Following a second round ofFM 4-64 loading (to compensate for dye release during acquisition of thesynaptolucin image, see FIG. 4B), the preparation was incubated on themicroscope stage with 20 M each of botulinum neurotoxin (BOPM serotypesB and F (39) plus 1 μM tetrodotoxin to suppress action potentials (andhence, dye release) during the incubation. After 3 hours of toxintreatment a second pair of images was recorded, and noted to differ fromthe first in two respects: i) the dimming of FM 4-64 fluorescence thatoriginally accompanied the hyperkalemic challenge now failed to occur,indicating that exocytosis was effectively blocked (compare FIG. 4B and4D), and ii) photon emissions from synaptolucin disappearedconcomitantly (compare FIGS. 4A and 4C). This ties the synaptolucinsignal firmly to the process of neurotransmitter release.

Estimates for the number of quanta released under the experimentalconditions described above and for the average observation time persynaptolucin can be derived by modelling vesicle release and recyclingas Poisson processes (1,2,41,42). At a typical hippocampal synapse, theprobability for exocytosis drops from an initial rate of about 20 quantasec⁻¹ (the “readily releasable pool”) to a basal rate of 2 quanta sec⁻¹(43); the transition between initial and basal release rates occursexponentially with a time constant of 1.2 sec (43). The probability ofrecycling is assumed constant throughout, with a t_(½) of 20 sec(12,44). When such a synapse is observed for 30 sec under maintainedhyperkalemic stimulation, an average of 67 quantal releases will takeplace, and the synaptolucins contained in one quantum (i.e., onevesicle) will emit for an average of 13 sec (see the legend to Table 1).Under the same conditions, an average of 12 photon registrations werecounted per synapse (Table 1). Correcting for the detection efficiency(see Table 1), this translates into about 312 photon emissions for theentire synapse, 4.7 photon emissions for a single vesicle, and a photonemission rate of 0.38 sec⁻¹ per vesicle, equalling that generated byabout three synaptolucin molecules in vitro at the same limitingluciferin concentration of 30 nM.

A fluctuation analysis (refs. 45, 46) of the photon counts in Table 1,obtained from the experiment shown in FIG. 2C, estimates the number ofreleased quanta as 51 per synapse and the photon emission rate as 0.49sec⁻¹ per vesicle, in rather close agreement with the values derivedfrom kinetic arguments alone (Table 1).

TABLE 1 Photons and Vesicles Photons Photon Counts per Field (Mean +/−SD) 12 +/− 3.9 Detection Efficiency 0.13 Collection Efficiency 0.29Overall Efficiency 0.04 Photon Emissions per Field 310 Vesicles Kineticsof Photon Count Method of Estimation Transmitter Release FluctuationsFusion Events 67 51 Photon Emissions per Vesicle 4.7 6.1 Photon EmissionRate per 0.38 0.49 Vesicle (sec⁻¹) Photons. The gray-level incrementcorresponding to a single photon count was determined from the histogramof FIG. 2C, and the number of photon registrations over a 30-sec periodcounted in 50 2 × 2-pixel fields, corresponding to 1.8 × 1.8 μm areas inthe specimen plane. These fields were selected by two criteria: i) FM4-64 fluorescence in excess of the 97^(th) percentile (see FIG. 2F), andii) a >5-fold increase in synaptolucin activity upon depolarization. Toconvert photon counts to photon emissions, two correction factors wereused: the detection efficiency of the intensifier photocathode(C2400-30H; Hamamatsu Photonics) at 462 nm, and the collectionefficiency of a 1.3 NA oil immersion objective, defined as the fractionof photon emissions from the focal plane that fall into the objective'sacceptance cone. Vesicles. Synaptic vesicle exocytosis and recyclingwere modeled as Poisson processes (1,2,41,42), using kinetic parametersobtained in studies of transmitter or dye release from identifiedsynapses of hippocampal neurons in culture (12,43,44). This stochasticmodel provided the basis for estimating the number of fusion events,either on the assumption of a fixed number of statistically independentrelease sites per synapse (41-43), or through an analysis of photoncount fluctuations (45,46).

Comparing the number of photon registrations (about 12 per synapse) withthe actual number of vesicle fusion events (about 60 per synapse)indicates that the majority of fusion events remained undetected withpresent technology. With a photon emission rate of 0.4-0.5 sec⁻¹ pervesicle and an overall photon detection efficiency of about 4% (Table1), the time between two successive photon registrations fromsynaptolucins originating in the same vesicle (the waiting time for thestochastic process) would average about 50 sec. This is considerablylonger than the average 13 sec for which a synaptolucin was observed.Hence under the conditions reported above, synaptolucins will often bere-internalized before a single photon emission can be detected, andthose vesicle fusion events that do register cannot be precisely locatedon a temporal scale.

Example 2 SynaptopHluorins

To generate pH-sensitive GFP mutants, a combination of directed andrandom strategies was employed. In the folded conformation of GFP, thechromophore is located in the core of the protein, shielded from directinteractions with the environment by a tight beta-barrel structure. Theremarkable stability of the fluorescent properties of wild-type proteinunder environmental perturbations, such as pH changes, is due to theprotected position of the chromophore. To allow GFP to function as a pHsensor, a mechanism had therefore to be found by which changes inexternal proton concentration could be relayed to and affect thechromophore.

The exemplified approach to converting GFP to a pH sensor involves aminoacid substitutions that couple changes in bulk pH to changes in theelectrostatic environment of the chromophore. To obtain suchsubstitutions, residues adjacent to 7 key positions were mutated. Thesekey positions are known from X-ray crystallography (Ref. 48, 55, 58) tobe part of the proton relay network of Tyr⁶⁶ (see FIG. 14A, 14B), and/orto alter the excitation spectrum when mutated (Ref. 47, 48, 59, 60). Keyresidues fulfilling one or both of these criteria include Gln-69,Gln-94, Arg-96, Asn-121, His-148, Phe-165, Ile-167, Gln-183, Thr-203,Ser-205, and Glu-222.

In the examples below, reversible spectral changes that would be gradedbetween pH 6 and 7 were sought (the pH in secretory vesicles (50, 51,53) is in the range of 5-6, while the extracellular pH is generally7.4), therefore the key residues themselves were not mutated, but ratherthe amino acids flanking them were altered. As in all beta-structures(GFP is an 11-stranded beta-barrel with a central alpha-helix carryingthe chromophore (55, 48, 58), FIG. 14A), the side chains of adjacentresidues alternate in orientation, such that those of amino acidsflanking key positions (whose side chains point towards the chromophore,FIG. 14B) face the surface of the protein and should thus be likely totitrate with bulk pH. Because histidine possesses the desired pK_(a)(ca. 6.4) for a sensor for exocytosis, histidine residues wereintroduced at positions 149, 151, 153, 164, 166, 168, 202, 204, and 206,singly or in combination. Changes in the charge of a critically located,outward-facing imidazole ring were expected to drive a spectral shift asa function of pH.

To this end, the coding region of GFP clone 10 (Ref. 47) was amplifiedby PCR, using the appropriate mutagenic oligonucleotides, ligated to theexpression vector pGEMEX2, and transformed into E.coli strain BL-21.Visibly fluorescent colonies were selected, and high-level expression ofGFP achieved by growing the clones to saturation in liquid culture, at25° C. and without IPTG induction. To prepare soluble extracts, cellsfrom 2-ml cultures were collected, washed, and resuspended in 50 mMTris/HCl, pH 8.0, containing 2 mM EDTA, 0.2 mg/ml lysozyme, and 20 μg/mlDNase I. After 2 hours on ice, lysates were clarified by centrifugationat 14,000 rpm for 15 min. Excitation spectra of lysates, diluted tenfoldinto 50 mM sodium cacodylate buffer, adjusted to pH 7.4 or 5.5 andcontaining 100 mM KCl, 1 mM MgCl₂, and 1 mM CaCl₂, were recorded in aPerkin-Elmer LS 50B spectrofluorimeter at room temperature. The emissionwavelength was set to 510 nm and the excitation wavelength scanned from360 to 500 nm, using 7.5-nm bandwidths for excitation and emission.

One of the candidate mutants generated in this first round ofmutagenesis, S202H, showed a 16% reduction in 395-nm excitation and a26% increase in 475-nm excitation, induced by a pH shift from 7.4 to 6.0(compare FIG. 8A and 8B). Clone S202H was subjected to further rounds ofmutagenesis in an effort to increase pH-responsiveness. Using degenerateoligonucleotides to incorporate more than one amino acid residue at eachposition, and to mutate more than one position simultaneously, cDNAregions including the key amino acid residues identified above, i.e.,codons for amino acid positions 94-97, 147-150, 164-168, 202-204, and221-223, were initially targeted for mutagenesis. In later mutagenesisexperiments, codons for amino acid positions 94-97, 146-149, 164-168,202-205, and 221-225, were mutated. The resulting libraries encompassedbetween 32 and 3,072 different nucleotide sequences in the initialexperiments, and between 20 and 8,000 different sequences in the laterexperiments. The PCR libraries were ligated to pGEMEX2, transformed intostrain BL-21, and visibly fluorescent colonies selected. Forhigh-throughput screening, liquid cultures were grown and lysatesprepared and analyzed in 96-well microtiter plates. For each well,fluorescence emitted at 510 nm after excitation at 400 and 460 nm wasrecorded at three pH values in a Labsystems Fluoroskan II fluorescentplate reader: first at pH 8.0 (lysis buffer), then after addition of 200mM sodium cacodylate to reduce the pH to 5.5, and finally after additionof 200 mM NaOH to revert the pH to 7.4. Fluorescence data weredigitized, corrected for volume changes due to buffer and NaOHadditions, and analyzed for changes in excitation peak ratios. Promisingcandidates were re-analyzed in detail, to obtain full excitation spectraand complete DNA sequences.

Including the later experiments, approximately 19,000 colonies werescreened altogether. After two rounds of mutagenesis, the processgenerated two distinct classes of pHluorins, which are referred to as“ratiometric” and “ecliptic” for the reasons discussed. These twoclasses were separately subjected to three and five additionalcombinatorial rounds, respectively, followed by one random round, ofmutagenesis. Finally, the two amino acid changes (V163A, S175G) known toimprove folding at 37° C. (ref.49) were introduced; these changes didnot affect the pHluorins' spectral properties but did increaseexpression levels at 37° C. The sequences and spectra of two prototypes,termed 1B11t and 14E12t, are shown in FIG. 8C and 8D and 22-23. Clone1B11t (See FIG. 22 for the amino acid sequence and 7A for the cDNAnucleic acid sequence) responded to a reduction in pH with a complete(and reversible) loss of fluorescence excitable at 475 nm, and an about8-fold reduction in fluorescence intensity excitable at 395 nm (FIG.8C). This type of mutant, which is referred to as an “ecliptic” pHluorinfor reasons discussed below, provides a preferred light-generatingmodule for the synaptopHluorins disclosed below. Clone 14E12t (see FIG.23 for the amino acid sequence and FIG. 7B for the cDNA nucleic acidsequence) responded to a reduction in pH with decreased fluorescenceexcitable at 395 nm, and increased fluorescence excitable at 475 nm(FIG. 8D). The class of “ratiometric” pHluorins exemplified by clones14E12 and 14E12t provides another preferred light-generating module forsynaptopHluorins.

The amino acid substitutions of the other clones are identified in Table2. Excitation spectra for the 1B11-like GFP mutants are shown in FIGS.9A-9D. Excitation spectra for the 14E12-like mutants is shown in FIGS.10A-10G. Corresponding cDNA nucleic acid sequences for these mutants areshown in FIGS. 11 and 12 respectively. The most preferred eclipticpHluorin is 8F3, and the most preferred ratiometric pHluorin is C6.

Although a limited number of mutations, in a limited number of regionsof the GFP protein are exemplified herein, it is anticipated thatmutations to other portions of the GFP protein may be made by themethods disclosed herein.

TABLE 2 Mutant Amino Acid Substitutions Amino Acid Substitutions inEcliptic pHluorins 1D10 S147D T161I S202H 2F10 S147D N149H T161I S202H2H2 S147D N149V T161I S202H 1B11 S147D N149Q T161I S202H 1B11t S147DN149Q T161I V163A S175G S202H 8F3 S147D N149Q T161I V163A S175G S202FQ204T A206T 8F6 S147D N149T T161I S202H 19E10 S147D N149L K166Q I167VR168H S202H Amino Acid Substitutions in Ratiometric pHluorins 14E12S147D N149D K166Q I167V S202H 14E12t S147D N149D V163A K166Q I167V S175GS202H 14C9 S147D N149L K166Q I167V S202H 14C8* S147P N149Y K166Q I167VR168H S202H 2G3 S147E N149L V163A R168H S175G S202H C6** S147E N149LV163A K166Q I167V R168H S175G S202H S202H T161I S202H 14D9 S147P N149WK166Q I167C R168H S202H 8H8 S147P N149W T161I S202H *Mutant 14C8 alsoincorporates G2S. *Mutant C6 also incorporates E132D, N164I, R168H, andL236V mutations.

Ratiometric pHluorins display a continuous and reversible excitationratio change between pH 7.5 and 5.5 (FIG. 14D), with a response time of<200 msec. Ecliptic pHluorins, by contrast, gradually lose fluorescenceintensity as pH is lowered, until at pH values of 6.0 and, theexcitation peak at 475 nm vanishes entirely (FIG. 14E). In anenvironment of pH<6.0, the protein is therefore invisible (eclipsed)under 475-nm excitation; however, it can still be seen (weakly) at 395nm. These changes are entirely reversible within <200 msec afterreturning to neutral pH.

For initial imaging experiments, ratiometric pHluorins were either

1) glycosylphosphatidylinositol (GPI)-anchored (61) at the cell surface(FIG. 15B),

2) inserted into the lumenal domain of TGN38 (62), an integral membraneprotein of the trans-Golgi network (TGN) (FIG. 15C), or

3) attached to the lumenally exposed C-terminus of cellubrevin (63), amembrane protein of the endosomal system (FIG. 15D).

Correct targeting of these constructs was confirmed

1) by the release of cell-bound fluorescence after GPI-anchor cleavage(61) with phosphatidylinositol-specific phospholipase C,

2) by co-localization with myc-tagged TGN38, or

3) by co-localization with internalized transferrin.

In all instances, the labelled subcellular compartment of transfectedHeLa cells was readily seen by wide-field fluorescence microscopy andcharacterized by a distinct 410/470-nm excitation ratio, R_(410/470)(FIG. 15). (The optimal excitation wavelengths for imaging were found tobe 410 and 470 nm rather than 395 and 475 nm. This probably reflects thedifferent optical trains of microscope and spectrofluorimeter as well asthe different fluorescence backgrounds in vivo and in vitro. Exposuretimes ranged from 50 to 200 msec per excitation wavelength, andR_(410/470) was sampled at up to 5 Hz.) The ratio remained stable within±3% during 2 minutes of continuous image acquisition at 1 Hz, indicatingthat the pHluorin did not photoisomerize (55) detectably at the lightintensities used.

To convert excitation ratios to pH values in this and other experiments,a standard curve was generated by imaging, in buffers of defined pH,cells expressing GPI-anchored pHluorin at their surface. Based on thisstandard curve (FIG. 15A), pH values (mean±SD) of 5.51±0.66 forendosomes (n=61) and 6.21±0.39 for the TGN (n=28) were determined,consistent with previous estimates of 5.0-5.5 and ca. 6.2, respectively.

Vacuolar (H⁺)-ATPases maintain the interior of synaptic vesicles athigher proton electrochemical potential than the cytoplasm, establishingan electrochemical gradient across the vesicle membrane that is tappedto concentrate neurotransmitter (51-53, 65). Whether this gradient ismainly electrical or chemical (i.e., a pH gradient) is determined by thevesicle membrane's anion conductances. The magnitudes of theseconductances, as well as the relative magnitudes of electrical andchemical potential in vivo, are unknown; reported estimates ofintravesicular pH concern purified synaptic vesicles in vitro (51-53).To perform a measurement in vivo, hippocampal neurons in low-densityculture (66) were infected with a herpes simplex virus (HSV) ampliconvector (67, 68) expressing a synaptopHluorin (69) in which the targetingmodule is the synaptic vesicle v-SNARE VAMP-2/synaptobrevin (65) and thelight-emitting unit is the ratiometric pHluorin, joined to VAMP at itslumenally exposed C-terminus. SynaptopHluorin fluorescence appeared inthe beads-on-a-string pattern typically seen when a single axon formsmultiple synapses with a single dendrite (FIG. 15E) and reported anintravesicular pH (mean±SD) of 5.67±0.71 (n=84).

Fusion of an acidified synaptic vesicle with the presynaptic membranewill establish continuity of its interior with the extracellular fluid,causing an essentially instantaneous rise of pH from ca. 5.7 to ca. 7.4.A synaptopHluorin, attached to the inner surface of the vesiclemembrane, will experience the change in pH and respond with a recordablechange of its excitation spectrum, which thus can serve as an index ofsynaptic activity. FIG. 16 illustrates this principle. FIG. 16A shows afield of neurites forming abundant synaptic contacts, revealed byimmunostaining for the synaptic vesicle protein synaptotagmin-I (65).Some of these contacts were made by neurons expressing the ratiometricsynaptopHluorin; they were easily distinguished by their greenfluorescence (compare the set of all synapses visible in FIG. 16A withthe subset of genetically tagged synapses visible in FIG. 16B).

The application of depolarizing solution containing 90 mM KCl elicited aprompt increase in R_(410/470) that could be recorded from many boutonsin parallel (FIG. 16C). The response depended on external Ca²⁺ andpeaked at 8-20% of the signal that would have resulted from simultaneousfusion of all of the vesicles in the bouton. The latter was estimated byneutralizing the pH in all synaptic vesicles with 50 mM NH₄Cl, whichreleases NH₃ that diffuses across cellular membranes and quenches freeprotons in acidified organelles (FIG. 16C). Relative to the maximumpossible response, the increase in R_(410/470) due to K⁺-depolarizationthus reflects a rapid rise in pH experienced by 8-20% of the vesicles ata synapse—a number that closely matches the size of the “readilyreleasable” pool (65, 70). This pool encompasses one to two dozen (70)of the 100-200 synaptic vesicles at an active zone (65) (i.e., 6-24%)and is released within the first two seconds after the onset of adepolarizing stimulus (70).

R_(410/470) remained elevated during continued depolarization,indicating that a steady state was attained in which continuedexocytosis was balanced by vesicle recycling, leaving a constantfraction of vesicle membrane protein externally disposed (FIG. 16C).After the depolarizing stimulus was withdrawn, R_(410/470) graduallyreturned to baseline (FIG. 16C). Vesicle membrane protein is known to bere-internalized by endocytosis to regenerate synaptic vesicles (with at_(½) of 10-20 seconds (71), in keeping with the time course observed inFIG. 16C), and synaptopHluorin is expected to return to its spectralbaseline as the vesicle acidifies.

The R_(410/470) value, whether it pertains to a single synapse or apopulation of many synapses in a region, therefore provides a runningaverage of synaptic activity during the previous several seconds. Likeall ratiometric indices, R_(410/470) is insensitive to variations inoptical path length, pHluorin concentration, and illumination intensity.These properties would make the ratiometric pHluorin an excellent probefor analyses of complex neural systems, when a large number of synapseshas to be surveyed in three dimensions and sampled serially to achievespatial resolution.

Ecliptic pHluorins, because they are non-fluorescent at pH<6 under470-nm excitation (FIG. 14E), eliminate fluorescence due to the largeexcess of resting vesicles and are thus potentially suited for detectingsingle vesicle fusion events. To test this, the mast cell line RBL-2H3,which releases histamine, serotonin, and other mediators when exocytosisis triggered (72), was chosen. When expressed in RBL-2H3 cells, eclipticsynaptopHluorin (a fusion protein of VAMP and the ecliptic pHluorin) waslocalized to scattered fluorescent puncta which were assumed to besecretory vesicles, and which under resting conditions were seen onlywith 410-nm and not with 470-nm excitation (FIG. 17A, frame 1). The pHin these granules, measured with the ratiometric pHluorin, averaged(mean±SD) 5.20±0.55 (n=29), the threshold for 470-nm excitation ofecliptic pHluorin.

After initiating a secretory response by cross-linkingsurface-receptor-bound IgE with an anti-IgE antibody (72), granulecontent was released into the medium (FIG. 17B), and changes influorescence excited at 470 nm occurred in locations harboring granules(FIG. 17A, frames 1-7). Individual spots of variable integratedintensity (the product of spot size and fluorescence intensity, FIG.17C) appeared suddenly, at various times after the stimulus to secrete(FIG. 17A, frames 2-7). The cumulative fluorescence of these individualevents closely followed the appearance of granule content in the medium(FIG. 17B), as expected if each event were due to fusion of a singlegranule (73), or of multiple granules undergoing compound exocytosis(73,74). In that case, the more intense events in FIG. 17C wouldcorrespond to compound exocytoses.

Occasionally, fluorescent spots disappeared, indicating granuleretrieval and re-acidification (follow the cluster marked by the arrowin frame 4 through frames 5 and 6). Within the time frame of theseexperiments (FIG. 17B), these “off” events (73) were less frequent thanthe “on” events of exocytosis, consistent with the slow resolution ofthe often tortuous membrane topology created at exocytosis sites (74).Rarely, a granule appeared, disappeared, and reappeared in what seemedto be the same location (arrows in frames 5-7). This could correspond tothe phenomenon of “flicker” (73), in which transient opening and closingof a fusion pore would cause the vesicle's internal pH (and thus, theecliptic pHluorin's emission intensity) to fluctuate.

Mutagenesis. Wild-type A. victoria gfp cDNA (pGFP-1; Clontech) wassubjected to PCR mutagenesis. Directed codon changes were introducedwith non-degenerate primers and Pwo polymerase; combinatorial stepsemployed primer libraries of 32- to 32,768-fold nucleotide degeneracyand Taq polymerase; random steps used low-fidelity amplification withTaq polymerase (7 mM MgCl₂, 0.5 mM MnCl₂, and a 5-fold excess of dCTPand dTTP over dGTP and dATP). PCR products were ligated to pGEMEX-2(Promega), transformed into E. coli, and visibly fluorescent colonieswere expanded for analysis. Clones were grown (at 25° C.; without IPTGinduction), lysed (on ice; in 50 mM Tris, pH 8.0, 2 mM EDTA, 0.2 mg/mllysozyme, 200 U/ml DNase I), and analyzed in 96-well plates. Threesuccessive readings of fluorescence emitted at 510 nm were obtained in aLabsystems Fluoroskan II plate reader equipped with 400- and 460-nmexcitation filters: the first at pH 8.0, the second after addition ofacid to reduce the pH to 6.0, and the third after addition of base torevert to pH 7.4. The plasmid encoding the mutant with the largestreversible, pH-dependent change in R_(410/460) served as the PCRtemplate in the next round of mutagenesis.

cDNAs encoding ratiometric and ecliptic pHluorins were sequenced andexpressed in pGEX-2T (Pharmacia). Fluorescence spectra were recorded onpure recombinant protein, obtained after thrombin cleavage of therespective GST fusion protein, in a Perkin-Elmer LS-50Bspectrofluorimeter. Response times to pH changes were estimated in timedrive mode, in a stirred cuvette that contained the pHluorin plusSNAFL-2 (Molecular Probes) as an internal standard.

Cells. GFP modules were linked to targeting modules via two-Ser-Gly-Gly-repeats and one -Thr-Gly-Gly- repeat; the latter containeda unique Age I site for insertion of GFP sequences. The insertion siteswere placed between signals for ER translocation and GPI-anchor addition(derived from preprolactin and decay accelerating factor (61),respectively), at the mature N-terminus of TGN38 (ref. 15), and at thevery C-termini of VAMP (65) and cellubrevin (63). All constructs carrieda single, lumenally exposed GFP module.

The vector pCI (Promega) was used to drive transient expression in HeLaand RBL-2H3 cells. Hippocampal neurons were infected with purifiedvirions of the quadruply deleted HSV strain (67) THZ.3 (α4⁻, α22⁻, α27⁻,U_(L)41⁻). Amplicon plasmids based on the pα4“a” backbone (68) werepackaged with the help of the complementing cell line (67) 7B, and theresulting mixture of vector and helper virions pelleted through 25%sucrose. Neural cultures were prepared as described (69), except thathippocampi were collected from embryonic (E19) rats, horse serum wasused, and 10 mM cytosine arabinoside was added from day 3 after plating.Experiments were performed after 17-29 days in vitro.

Microscopy. Two days after transfection or viral infection, cells weretransferred to imaging buffer (25 mM Na-Hepes, pH 7.4, 119 mM NaCl, 2.5mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 30 mM glucose) and at 37° C. imaged on aZeiss Axiovert microscope equipped with a 40×, 1.3 NA PLAN-NEOFLUARobjective and 1.6× and 2.5×OPTOVAR inserts. Experiments were controlledthrough METAFLUOR 3.0 (Universal Imaging), with off-line backgroundsubtraction and image analysis, using METAMORPH 3.0 (Universal Imaging)and MATHEMATICA 3.0 (Wolfram Research). To rapidly alternate betweennarrow excitation bands, a POLYCHROME II grating monochromator (TillPhotonics; 75 W xenon lamp, 12-nm bandwidth) was coupled into theepi-illumination port of the microscope. Emitted light was passedthrough a dichromatic mirror (500DCXR) and a bandpass filter (HQ535/50,both from Chroma Technologies) and collected on a PENTAMAX-512EFTframe-transfer camera with fiber coupled GEN IV image intensifier(Princeton Instruments; cooled 12-bit EEV-37 CCD array).

While we have described herein a number of embodiments of the invention,it is apparent that the basic constructions can be altered to provideother embodiments which utilize the methods of this invention.Therefore, it will be appreciated that the scope of this invention isdefined by the claims appended hereto rather than by the specificembodiments which have been presented hereinbefore by way of example.

REFERENCES

The following publications are incorporated herein by reference in theirentirety:

1. Redman, S. (1990) Physiol. Rev. 70:165-198.

2. Stevens, C. F. & Wang, Y. (1995) Neuron 14:795-802.

3. Anderson, J. A. & Rosenfeld, E., eds. (1988) Neurocomputing (NUTPress, Cambridge).

4. Shepherd, G. M., ed. (1990) The Synaptic Organization of the Brain(Oxford University Press, Oxford), 3rd Ed.

5. McKenna, T., Davis, J. & Zornetzer, S. E., eds. (1992) Single NeuronComputation (Academic Press, Boston).

6. Meister, M., Pine, J. & Baylor D. A. (1994) J. Neurosci. Meth.51:95-106.

7. Stenger, D. A. & McKenna, T. M., eds. (1994) Enabling Technologiesfor Cultured Neural Networks (Academic Press, San Diego).

8. Grinvald, A. (1985) Annu. Rev. Neurosci. 8:263-305.

9. Tsien, R. Y. (1989) Annu. Rev. Neurosci. 12:227-253.

10. Tsien, R. Y. & Waggoner, A. (1995) in Handbook of BiologicalConfocal Microscopy, ed. Pawley, J. B. (Plenum, New York), 2nd Ed., pp.267-279.

11. Betz, W. J. & Bewick, G. S. (1992) Science 255:200-203.

12. Ryan, T. A., Reuter, H., Wendland, B., Schweizer, F., Tsien, R. W. &Smith, S. J. (1993) Neuron 11:713-724.

13. Meister, M. (1996) Proc. Natl. Acad. Sci. USA 93:609-614.

14. Katz, L. C. & Shatz, C. J. (1996) Science 274:1133-1138.

15. Kuypers, H. G. J. M. & Ugolini, G. (1990) Trends Neurosci. 13:71-75.

16. Thompson, E. M., Nagata, S. & Tsuji, F. I. (1989) Proc. Natl. Acad.Sci. USA 86:6567-6571.

17. Ho, D. Y. (1994) Methods Cell Biol. 43:191-210.

18. Lawrence, M. S., Ho, D. Y., Dash, R. & Sapolsky, R. M. (1995) Proc.Natl. Acad. Sci. USA 92:7247-7251.

19. Perin, M. S., Fried, V. A., Mignery, G. A., Jahn, R. & Suldhof, T.C. (1990) Nature 345:260-263.

20. Elferink, L. A., Trimble, W. S. & Scheller, R. H. (1989) J. Biol.Chem. 264:11061-11064.

21. Sudhof, T. C., Baumert, M., Perin, M. S. & Jahn, R. (1989) Neuron2:1475-1481.

22. DeLuca, N. A., McCarthy, A. M. & Schaffer, P. A. (1984) J. Virol.56:558-5570.

23. Matthew, W. D., Tsavaler, L. & Reichardt, L. F. (1981) J. Cell Biol.91:257-269.

24. Edelmann, L., Hanson, P. I., Chapman, E. R. & Jahn, R. (1995) EMBOJ. 14:224-231.

25. Clift-O'Grady, L., Linstedt, A. D., Lowe, A. W., Grote, E. & Kelly,R. B. (1990) J. Cell Biol. 110:1693-1703.

26. Hastings, J. W. & Weber, G. (1963) J. Opt. Soc. Am. 53:1410-1415.

27. Huettner, W. J. & Baughman, R. W. (1986) J. Neurosci. 6:3044-3060.

28. Geppert, M., Goda, Y., Hammer, R. E., Li, C., Rosahl, T. W.,Stevens, C. F. & Sudhof, T. C. (1994) Cell 79:717-727.

29. Johnson, F. H. & Shimomura, O. (1978) Methods Enzymol. 57:331-364.

30. Shimomura, O., Johnson, F. H. & Saiga, Y. (1961) J. Cell. Comp.Physiol. 58:113-124.

31. Inouye, S., Ohmiya, Y., Toya, Y. & Tsuji, F. J. (1992) Proc. Natl.Acad. Sci. USA 89:9584-9587.

32. Kishi, Y., Goto, T., Hirata, Y., Shimomura, O. & Johnson, F. H.(1966) Tetrahedron Lett. 29:3427-3436.

33. Shimomura, O., Johnson, F. H. & Masugi, T. (1969) Science164:1299-1300.

34. Shimomura, O. & Johnson, F. H. (1970) Photochem. Photobiol.12:291-295.

35. Wulff, K. (1981) in Bioluminescence and Chemiluminescence, DeLuca,M. A. & McElroy, W. D., eds. (Academic Press, New York), p. 219.

36. Henkel, A. W. & Betz, W. J. (1995) J. Neurosci. 15:8246-8258.

37. Feany, M. B., Yee, A. G., Delvy M. L. & Buckley, K. M. (1993) J.Cell Biol. 123:575-584.

38. Duda, R. O. & Hart, P. E. (1973) Pattern Classification and SceneAnalysis (John Wiley & Sons, New York).

39. Montecucco, C. & Schiavo, G. (1995) Q. Rev. Biophys. 28:423-472.

40. Bliss, T. V. P. & Collingridge, G. L. (1993) Nature 361:31-39.

41. Katz, B. (1969) The Release of Neural Transmitter Substances(Liverpool University Press, Liverpool).

42. Bekkers, J. M. & Stevens, C. F. (1995) J. Neurophysiol.73:1145-1156.

43. Stevens, C. F. & Tsujimoto, T. (1995) Proc. Natl. Acad. Sci. USA92:846-849.

44. Ryan, T. A., Smith, S. J. & Reuter, H. (1996) Proc. Natl. Acad. Sci.USA 93:5567-5571.

45. Katz, B. & Miledi, R. (1972) J. Physiol. 224:665-699.

46. Neher, E. & Stevens, C. F. (1977) Annu. Rev. Biophys. Bioeng.6:345-381.

47. Heim, R., Prasher, D. C., Tsien, R. Y. (1994) Proc. Natl. Acad. Sci.USA 91:12501-12504.

48. Ormö, M., Cubitt, A. B., Kalio, K., Gross, L. A., Tsien, R. Y.,Remington, S. J. (1996) Science 273: 1392-1395.

49. Siemering, K. R., Golbik, R., Sever, R., Haseloff, J. (1996) Curr.Biol. 6:1653-1663.

50. Anderson, R. G. & Orci, L. A view of acidic intracellularcompartments. J Cell Biol 106, 539-43 (1988).

51. Füldner, H. H. & Stadler, H. ³¹P-NMR analysis of synaptic vesicles:status of ATP and internal pH. Eur J Biochem 121, 519-24 (1982).

52. Maycox, P. R., Hell, J. W. & Jahn, R. Amino acid neurotransmission:spotlight on synaptic vesicles. Trends Neurosci 13, 83-7 (1990).

53. Tabb, J. S., Kish, P. E., Van Dyke, R. & Ueda, T. Glutamatetransport into synaptic vesicles. Roles of membrane potential, pHgradient, and intravesicular pH. J Biol Chem 267, 15412-8 (1992).

54. Ward, W. W. in Bioluminescence and chemiluminescence. (eds. DeLuca,M. A. & McElroy, W. D.) 235-42 (Academic Press, New York, 1981).

55. Brejc, K., et al. Structural basis for dual excitation andphotoisomerization of the Aequorea victoria green fluorescent protein.Proc Natl Acad Sci USA 94, 2306-11 (1997).

56. Chattoraj, M., King, B. A., Bublitz, G. U. & Boxer, S. Ultra-fastexcited state dynamics in green fluorescent protein: Multiple states andproton transfer. Proc Natl Acad Sci USA 93, 8362-8367 (1996).

57. Heim, R., Prasher, D. C. & Tsien, R. Y. Wavelength mutations andposttranslational autoxidation of green fluorescent protein. Proc NatlAcad Sci USA 91, 12501-4 (1994).

58. Yang, F., Moss, L. G. & Phillips, G. N., Jr. The molecular structureof green fluorescent protein. Nature Biotech. 14, 1246-1251 (1996).

59. Ehrig, T., O'Kane, D. J. & Prendergast, F. G. Green-fluorescentprotein mutants with altered fluorescence excitation spectra. FEBS Lett367, 163-6 (1995).

60. Heim, R. & Tsien, R. Y. Engineering green fluorescent protein forimproved brightness, longer wavelengths and fluorescence resonanceenergy transfer. Curr Biol 6, 178-82 (1996).

61. Caras, I. W., Weddell, G. N., Davitz, M. A., Nussenzweig, V. &Martin, D. W., Jr. Signal for attachment of a phospholipid membraneanchor in decay accelerating factor. Science 238, 1280-3 (1987).

62. Luzio, J. P., et al. Identification, sequencing and expression of anintegral membrane protein of the trans-Golgi network (TGN38). Biochem J270, 97-102 (1990).

63. McMahon, H. T., et al. Cellubrevin is a ubiqitous tetanus-toxinsubstrate homologous to a putative synaptic vesicle fusion protein.Nature 364, 346-49 (1993).

64. Seksek, O., Biwersi, J. & Verkman, A. S. Direct measurement oftrans-Golgi pH in living cells and regulation by second messengers. J.Biol. Chem. 270, 4967-70 (1995).

65. Sudhof, T. C. The synaptic vesicle cycle: a cascade ofprotein-protein interactions. Nature 375, 645-53 (1995).

66. Goslin, K. & Banker, G. in Culturing nerve cells (eds. Banker, G. &Goslin, K.) 251-81 (NET Press, Cambridge, 1991).

67. Marconi, P., et al. Replication-defective herpes simplex virusvectors for gene transfer in vivo. Proc Natl Acad Sci USA 93, 11319-20(1996).

68. Lawrence, M. S., Ho, D. Y., Dash, R. & Sapolsky, R. M. Herpessimplex virus vectors overexpressing the glucose transporter geneprotect against seizure-induced neuron loss. Proc Natl Acad Sci USA 92,7247-51 (1995).

69. Miesenbock, G. & Rothman, J. E. Patterns of synaptic activity inneural networks recorded by light emission from synaptolucins. Proc.Natl. Acad. Sci. USA 94, 3402-3407 (1997).

70. Stevens, C. F. & Tsujimoto, T. Estimates for the pool size ofreleasable quanta at a single central synapse and for the time requiredto refill the pool. Proc Natl Acad Sci USA 92, 846-9 (1995).

71. Ryan, T. A. Endocytosis at nerve terminals: timing is everything.Neuron 17, 1035-7 (1996).

72. Roa, M., Paumet, F., Le Mao, J., David, B. & Blank, U. Involvementof the ras-like GTPase rab3d in RBL-2H3 mast cell exocytosis followingstimulation via high affinity IgE receptors (FcERI). J Immunol 159,2815-23 (1997).

73. Fernandez, J. M., Neher, E. & Gomperts, B. D. Capacitancemeasurements reveal stepwise fusion events in degranulating mast cells.Nature 312, 453-5 (1984).

74. Chandler, D. E. & Heuser, J. E. Arrest of membrane fusion events inmast cells by quick-freezing. J Cell Biol 86, 666-74 (1980).

75. Ullrich, A. & Schlessinger, J. Signal transduction by receptors withtyrosine kinase activity. Cell 61, 203-12 (1990).

76. Yu, S. S., Lefkowitz, R. J. & Hausdorff, W. P. β-adrenergic receptorsequestration: A potential mechanism of receptor resensitization. J BiolChem 268, 337-41 (1993).

77. James, D. E. & Piper, R. C. Insulin resistance, diabetes, and theinsulin-regulated trafficking of GLUT-4. J Cell Biol 126, 1123-6 (1994).

39 242 AMINO ACID UNKNOWN LINEAR PROTEIN GREEN FLUORESCENT PROTEIN 1 MetGly Lys Gly Glu Glu Leu Phe Thr Gly Val Val 1 5 10 Pro Ile Leu Val GluLeu Asp Gly Asp Val Asn Gly 15 20 His Lys Phe Ser Val Ser Gly Glu GlyGlu Gly Asp 25 30 35 Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 4045 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 50 55 60 Val Thr ThrPhe Ser Tyr Gly Val Gln Cys Phe Ser 65 70 Arg Tyr Pro Asp His Met LysArg His Asp Phe Phe 75 80 Lys Ser Ala Met Pro Glu Gly Tyr Val Gln GluArg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 100 105 ArgAla Glu Val Lys Phe Glu Gly Asp Thr Leu Val 110 115 120 Asn Arg Ile GluLeu Lys Gly Ile Asp Phe Lys Glu 125 130 Asp Gly Asn Ile Leu Gly His LysLeu Glu Tyr Asn 135 140 Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys145 150 155 Gln Lys Asn Gly Thr Lys Val Asn Phe Lys Ile Arg 160 165 HisAsn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp 170 175 180 His Tyr Gln GlnAsn Thr Pro Ile Gly Asp Gly Pro 185 190 Val Leu Leu Pro Asp Asn His TyrLeu Ser Thr Gln 195 200 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp205 210 215 His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly 220 225 IleThr His Gly Met Asp Glu Leu Tyr Lys Ser Gly 230 235 240 Ser Arg 729NUCLEIC ACID UNKNOWN UNKNOWN 2 ATGGGTAAAG GAGAAGAACT TTTCACTGGAGTTGTCCCAA 40 TTCTTGTTGA ATTAGATGGT GATGTTAATG GGCACAAATT 80 TTCTGTCAGTGGAGAGGGTG AAGGTGATGC AACATACGGA 120 AAACTTACCC TTAAATTTAT TTGCACTACTGGAAAACTAC 160 CTGTTCCATG GCCAACACTT GTCACTACTT TCTCTTATGG 200TGTTCAATGC TTTTCAAGAT ACCCAGATCA TATGAAACGG 240 CATGACTTTT TCAAGAGTGCCATGCCCGAA GGTTATGTAC 280 AGGAAAGAAC TATATTTTTC AAAGATGACG GGAACTACAA320 GACACGTGCT GAAGTCAAGT TTGAAGGTGA TACCCTTGTT 360 AATAGAATCGAGTTAAAAGG TATTGATTTT AAAGAAGATG 400 GAAACATTCT TGGACACAAA TTGGAATACAACTATAACTC 440 ACACAATGTA TACATCATGG CAGACAAACA AAAGAATGGA 480ACCAAAGTTA ACTTCAAAAT TAGACACAAC ATTGAAGATG 520 GAAGCGTTCA ACTAGCAGACCATTATCAAC AAAATACTCC 560 AATTGGCGAT GGCCCTGTCC TTTTACCAGA CAACCATTAC600 CTGTCCACAC AATCTGCCCT TTCGAAAGAT CCCAACGAAA 640 AGAGAGACCACATGGTCCTT CTTGAGTTTG TAACAGCTGC 680 TGGGATTACA CATGGCATGG ATGAACTATACAAGTCCGGA 720 TCTAGATAA 729 717 NUCLEIC ACID UNKNOWN UNKNOWN 3ATGAGTAAAG GAGAAGAACT TTTCACTGGA GTTGTCCCAA 40 TTCTTGTTGA ATTAGATGGTGATGTTAATG GGCACAAATT 80 TTCTGTCAGT GGAGAGGGTG AAGGTGATGC AACATACGGA 120AAACTTACCC TTAAATTTAT TTGCACTACT GGAAAACTAC 160 CTGTTCCATG GCCAACACTTGTCACTACTT TCTCTTATGG 200 TGTTCAATGC TTTTCAAGAT ACCCAGATCA TATGAAACGG240 CATGACTTTT TCAAGAGTGC CATGCCCGAA GGTTATGTAC 280 AGGAAAGAACTATATTTTTC AAAGATGACG GGAACTACAA 320 GACACGTGCT GAAGTCAAGT TTGAAGGTGATACCCTTGTT 360 AATAGAATCG AGTTAAAAGG TATTGATTTT AAAGAAGATG 400GAAACATTCT TGGACACAAA TTGGAATACA ACTATAACGA 440 TCACCAGGTG TACATCATGGCAGACAAACA AAAGAATGGA 480 ATCAAAGCTA ACTTCAAAAT TAGACACAAC ATTGAAGATG520 GAGGCGTTCA ACTAGCAGAC CATTATCAAC AAAATACTCC 560 AATTGGCGATGGCCCTGTCC TTTTACCAGA CAACCATTAC 600 CTGCACACAC AATCTGCCCT TTCGAAAGATCCCAACGAAA 640 AGAGAGACCA CATGGTCCTT CTTGAGTTTG TAACAGCTGC 680TGGGATTACA CATGGCATGG ATGAACTATA CAAATAA 717 717 NUCLEIC ACID UNKNOWNUNKNOWN 14E12t 4 ATGAGTAAAG GAGAAGAACT TTTCACTGGA GTTGTCCCAA 40TTCTTGTTGA ATTAGATGGT GATGTTAATG GGCACAAATT 80 TTCTGTCAGT GGAGAGGGTGAAGGTGATGC AACATACGGA 120 AAACTTACCC TTAAATTTAT TTGCACTACT GGAAAACTAC160 CTGTTCCATG GCCAACACTT GTCACTACTT TCTCTTATGG 200 TGTTCAATGCTTTTCAAGAT ACCCAGATCA TATGAAACGG 240 CATGACTTTT TCAAGAGTGC CATGCCCGAAGGTTATGTAC 280 AGGAAAGAAC TATATTTTTC AAAGATGACG GGAACTACAA 320GACACGTGCT GAAGTCAAGT TTGAAGGTGA TACCCTTGTT 360 AATAGAATTG AGTTAAAAGGTATTGATTTT AAAGAAGATG 400 GAAACATTCT TGGACACAAA TTGGAGTACA ACTATAACGA440 TCACGATGTG TACATCATGG CAGACAAACA AAAGAATGGT 480 ACCAAAGCTAACTTTCAAGT TCGCCACAAC ATTGAAGATG 520 GAGGCGTTCA ACTAGCAGAC CATTATCAACAAAATACTCC 560 AATTGGCGAT GGCCCTGTCC TTTTACCAGA CAACCATTAC 600CTGCACACAC AATCTGCCCT TTCGAAAGAT CCCAACGAAA 640 AGAGAGACCA CATGGTCCTTCTTGAGTTTG TAACAGCTGC 680 TGGGATTACA CATGGCATGG ATGAACTATA CAAATAA 717717 NUCLEIC ACID UNKNOWN UNKNOWN 1D10 5 ATGAGTAAAG GAGAAGAACT TTTCACTGGAGTTGTCCCAA 40 TTCTTGTTGA ATTAGATGGT GATGTTAATG GGCACAAATT 80 TTCTGTCAGTGGAGAGGGTG AAGGTGATGC AACATACGGA 120 AAACTTACCC TTAAATTTAT TTGCACTACTGGAAAACTAC 160 CTGTTCCATG GCCAACACTT GTCACTACTT TCTCTTATGG 200TGTTCAATGC TTTTCAAGAT ACCCAGATCA TATGAAACGG 240 CATGACTTTT TCAAGAGTGCCATGCCCGAA GGTTATGTAC 280 AGGAAAGAAC TATATTTTTC AAAGATGACG GGAACTACAA320 GACACGTGCT GAAGTCAAGT TTGAAGGTGA TACCCTTGTT 360 AATAGAATCGAGTTAAAAGG TATTGATTTT AAAGAAGATG 400 GAAACATTCT TGGACACAAA TTGGAATACAACTATAACGA 440 TCACAATGTG TACATCATGG CAGACAAACA AAAGAATGGA 480ATCAAAGTTA ACTTCAAAAT TAGACACAAC ATTGAAGATG 520 GAAGCGTTCA ACTAGCAGACCATTATCAAC AAAATACTCC 560 AATTGGCGAT GGCCCTGTCC TTTTACCAGA CAACCATTAC600 CTGCACACAC AATCTGCCCT TTCGAAAGAT CCCAACGAAA 640 AGAGAGACCACATGGTCCTT CTTGAGTTTG TAACAGCTGC 680 TGGGATTACA CATGGCATGG ATGAACTATACAAATAA 717 717 NUCLEIC ACID UNKNOWN UNKNOWN 2F10 6 ATGAGTAAAGGAGAAGAACT TTTCACTGGA GTTGTCCCAA 40 TTCTTGTTGA ATTAGATGGT GATGTTAATGGGCACAAATT 80 TTCTGTCAGT GGAGAGGGTG AAGGTGATGC AACATACGGA 120 AAACTTACCCTTAAATTTAT TTGCACTACT GGAAAACTAC 160 CTGTTCCATG GCCAACACTT GTCACTACTTTCTCTTATGG 200 TGTTCAATGC TTTTCAAGAT ACCCAGATCA TATGAAACGG 240CATGACTTTT TCAAGAGTGC CATGCCCGAA GGTTATGTAC 280 AGGAAAGAAC TATATTTTTCAAAGATGACG GGAACTACAA 320 GACACGTGCT GAAGTCAAGT TTGAAGGTGA TACCCTTGTT360 AATAGAATCG AGTTAAAAGG TATTGATTTT AAAGAAGATG 400 GAAACATTCTTGGACACAAA TTGGAATACA ACTATAACGA 440 TCACCATGTG TACATCATGG CAGACAAACAAAAGAATGGA 480 ATCAAAGTTA ACTTCAAAAT TAGACACAAC ATTGAAGATG 520GAAGCGTTCA ACTAGCAGAC CATTATCAAC AAAATACTCC 560 AATTGGCGAT GGCCCTGTCCTTTTACCAGA CAACCATTAC 600 CTGCACACAC AATCTGCCCT TTCGAAAGAT CCCAACGAAA640 AGAGAGACCA CATGGTCCTT CTTGAGTTTG TAACAGCTGC 680 TGGGATTACACATGGCATGG ATGAACTATA CAAATAA 717 717 NUCLEIC ACID UNKNOWN UNKNOWN 2H2 7ATGAGTAAAG GAGAAGAACT TTTCACTGGA GTTGTCCCAA 40 TTCTTGTTGA ATTAGATGGTGATGTTAATG GGCACAAATT 80 TTCTGTCAGT GGAGAGGGTG AAGGTGATGC AACATACGGA 120AAACTTACCC TTAAATTTAT TTGCACTACT GGAAAACTAC 160 CTGTTCCATG GCCAACACTTGTCACTACTT TCTCTTATGG 200 TGTTCAATGC TTTTCAAGAT ACCCAGATCA TATGAAACGG240 CATGACTTTT TCAAGAGTGC CATGCCCGAA GGTTATGTAC 280 AGGAAAGAACTATATTTTTC AAAGATGACG GGAACTACAA 320 GACACGTGCT GAAGTCAAGT TTGAAGGTGATACCCTTGTT 360 AATAGAATCG AGTTAAAAGG TATTGATTTT AAAGAAGATG 400GAAACATTCT TGGACACAAA TTGGAATACA ACTATAACGA 440 TCACGTGGTG TACATCATGGCAGACAAACA AAAGAATGGA 480 ATCAAAGTTA ACTTCAAAAT TAGACACAAC ATTGAAGATG520 GAAGCGTTCA ACTAGCAGAC CATTATCAAC AAAATACTCC 560 AATTGGCGATGGCCCTGTCC TTTTACCAGA CAACCATTAC 600 CTGCACACAC AATCTGCCCT TTCGAAAGATCCCAACGAAA 640 AGAGAGACCA CATGGTCCTT CTTGAGTTTG TAACAGCTGC 680TGGGATTACA CATGGCATGG ATGAACTATA CAAATAA 717 717 NUCLEIC ACID UNKNOWNUNKNOWN 1B11 8 ATGAGTAAAG GAGAAGAACT TTTCACTGGA GTTGTCCCAA 40 TTCTTGTTGAATTAGATGGT GATGTTAATG GGCACAAATT 80 TTCTGTCAGT GGAGAGGGTG AAGGTGATGCAACATACGGA 120 AAACTTACCC TTAAATTTAT TTGCACTACT GGAAAACTAC 160CTGTTCCATG GCCAACACTT GTCACTACTT TCTCTTATGG 200 TGTTCAATGC TTTTCAAGATACCCAGATCA TATGAAACGG 240 CATGACTTTT TCAAGAGTGC CATGCCCGAA GGTTATGTAC280 AGGAAAGAAC TATATTTTTC AAAGATGACG GGAACTACAA 320 GACACGTGCTGAAGTCAAGT TTGAAGGTGA TACCCTTGTT 360 AATAGAATCG AGTTAAAAGG TATTGATTTTAAAGAAGATG 400 GAAACATTCT TGGACACAAA TTGGAATACA ACTATAACGA 440TCACCAGGTG TACATCATGG CAGACAAACA AAAGAATGGA 480 ATCAAAGTTA ACTTCAAAATTAGACACAAC ATTGAAGATG 520 GAAGCGTTCA ACTAGCAGAC CATTATCAAC AAAATACTCC560 AATTGGCGAT GGCCCTGTCC TTTTACCAGA CAACCATTAC 600 CTGCACACACAATCTGCCCT TTCGAAAGAT CCCAACGAAA 640 AGAGAGACCA CATGGTCCTT CTTGAGTTTGTAACAGCTGC 680 TGGGATTACA CATGGCATGG ATGAACTATA CAAATAA 717 717 NUCLEICACID UNKNOWN UNKNOWN 8F6 9 ATGAGTAAAG GAGAAGAACT TTTCACTGGA GTTGTCCCAA40 TTCTTGTTGA ATTAGATGGT GATGTTAATG GGCACAAATT 80 TTCTGTCAGT GGAGAGGGTGAAGGTGATGC AACATACGGA 120 AAACTTACCC TTAAATTTAT TTGCACTACT GGAAAACTAC160 CTGTTCCATG GCCAACACTT GTCACTACTT TCTCTTATGG 200 TGTTCAATGCTTTTCAAGAT ACCCAGATCA TATGAAACGG 240 CATGACTTTT TCAAGAGTGC CATGCCCGAAGGTTATGTAC 280 AGGAAAGAAC TATATTTTTC AAAGATGACG GGAACTACAA 320GACACGTGCT GAAGTCAAGT TTGAAGGTGA TACCCTTGTT 360 AATAGAATCG AGTTAAAAGGTATTGATTTT AAAGAAGATG 400 GAAACATTCT TGGACACAAA TTGGAATACA ACTATAACGA440 TCACACTGTG TACATCATGG CAGACAAACA AAAGAATGGA 480 ATCAAAGTTAACTTCAAAAT TAGACACAAC ATTGAAGATG 520 GAAGCGTTCA ACTAGCAGAC CATTATCAACAAAATACTCC 560 AATTGGCGAT GGCCCTGTCC TTTTACCAGA CAACCATTAC 600CTGCACACAC AATCTGCCCT TTCGAAAGAT CCCAACGAAA 640 AGAGAGACCA CATGGTCCTTCTTGAGTTTG TAACAGCTGC 680 TGGGATTACA CATGGCATGG ATGAACTATA CAAATAA 717717 NUCLEIC ACID UNKNOWN UNKNOWN 19E10 10 ATGAGTAAAG GAGAAGAACTTTTCACTGGA GTTGTCCCAA 40 TTCTTGTTGA ATTAGATGGT GATGTTAATG GGCACAAATT 80TTCTGTCAGT GGAGAGGGTG AAGGTGATGC AACATACGGA 120 AAACTTACCC TTAAATTTATTTGCACTACT GGAAAACTAC 160 CTGTTCCATG GCCAACACTT GTCACTACTT TCTCTTATGG200 TGTTCAATGC TTTTCAAGAT ACCCAGATCA TATGAAACGG 240 CATGACTTTTTCAAGAGTGC CATGCCCGAA GGTTATGTAC 280 AGGAAAGAAC TATATTTTTC AAAGATGACGGGAACTACAA 320 GACACGTGCT GAAGTCAAGT TTGAAGGTGA TACCCTTGTT 360AATAGAATTG AGTTAAAAGG TATTGATTTT AAAGAAGATG 400 GAAACATTCT TGGACACAAATTGGAGTACA ACTATAACGA 440 TCACTTGGTG TACATCATGG CAGACAAACA AAAGAATGGT480 ACCAAAGTTA ACTTTCAAGT TCACCACAAC ATTGAAGATG 520 GAAGCGTTCAACTAGCAGAC CATTATCAAC AAAATACTCC 560 AATTGGCGAT GGCCCTGTCC TTTTACCAGACAACCATTAC 600 CTGCACACAC AATCTGCCCT TTCGAAAGAT CCCAACGAAA 640AGAGAGACCA CATGGTCCTT CTTGAGTTTG TAACAGCTGC 680 TGGGATTACA CATGGCATGGATGAACTATA CAAATAA 717 717 NUCLEIC ACID UNKNOWN UNKNOWN 14E12 11ATGAGTAAAG GAGAAGAACT TTTCACTGGA GTTGTCCCAA 40 TTCTTGTTGA ATTAGATGGTGATGTTAATG GGCACAAATT 80 TTCTGTCAGT GGAGAGGGTG AAGGTGATGC AACATACGGA 120AAACTTACCC TTAAATTTAT TTGCACTACT GGAAAACTAC 160 CTGTTCCATG GCCAACACTTGTCACTACTT TCTCTTATGG 200 TGTTCAATGC TTTTCAAGAT ACCCAGATCA TATGAAACGG240 CATGACTTTT TCAAGAGTGC CATGCCCGAA GGTTATGTAC 280 AGGAAAGAACTATATTTTTC AAAGATGACG GGAACTACAA 320 GACACGTGCT GAAGTCAAGT TTGAAGGTGATACCCTTGTT 360 AATAGAATTG AGTTAAAAGG TATTGATTTT AAAGAAGATG 400GAAACATTCT TGGACACAAA TTGGAGTACA ACTATAACGA 440 TCACGATGTG TACATCATGGCAGACAAACA AAAGAATGGT 480 ACCAAAGTTA ACTTTCAAGT TCGCCACAAC ATTGAAGATG520 GAAGCGTTCA ACTAGCAGAC CATTATCAAC AAAATACTCC 560 AATTGGCGATGGCCCTGTCC TTTTACCAGA CAACCATTAC 600 CTGCACACAC AATCTGCCCT TTCGAAAGATCCCAACGAAA 640 AGAGAGACCA CATGGTCCTT CTTGAGTTTG TAACAGCTGC 680TGGGATTACA CATGGCATGG ATGAACTATA CAAATAA 717 717 NUCLEIC ACID UNKNOWNUNKNOWN 14C9 12 ATGAGTAAAG GAGAAGAACT TTTCACTGGA GTTGTCCCAA 40TTCTTGTTGA ATTAGATGGT GATGTTAATG GGCACAAATT 80 TTCTGTCAGT GGAGAGGGTGAAGGTGATGC AACATACGGA 120 AAACTTACCC TTAAATTTAT TTGCACTACT GGAAAACTAC160 CTGTTCCATG GCCAACACTT GTCACTACTT TCTCTTATGG 200 TGTTCAATGCTTTTCAAGAT ACCCAGATCA TATGAAACGG 240 CATGACTTTT TCAAGAGTGC CATGCCCGAAGGTTATGTAC 280 AGGAAAGAAC TATATTTTTC AAAGATGACG GGAACTACAA 320GACACGTGCT GAAGTCAAGT TTGAAGGTGA TACCCTTGTT 360 AATAGAATTG AGTTAAAAGGTATTGATTTT AAAGAAGATG 400 GAAACATTCT TGGACACAAA TTGGAGTACA ACTATAACGA440 TCACCTGGTG TACATCATGG CAGACAAACA AAAGAATGGT 480 ACCAAAGTTAACTTTCAAGT TCGCCACAAC ATTGAAGATG 520 GAAGCGTTCA ACTAGCAGAC CATTATCAACAAAATACTCC 560 AATTGGCGAT GGCCCTGTCC TTTTACCAGA CAACCATTAC 600CTGCACACAC AATCTGCCCT TTCGAAAGAT CCCAACGAAA 640 AGAGAGACCA CATGGTCCTTCTTGAGTTTG TAACAGCTGC 680 TGGGATTACA CATGGCATGG ATGAACTATA CAAATAA 717717 NUCLEIC ACID UNKNOWN UNKNOWN 14C8 13 ATGAGTAAAG GAGAAGAACTTTTCACTGGA GTTGTCCCAA 40 TTCTTGTTGA ATTAGATGGT GATGTTAATG GGCACAAATT 80TTCTGTCAGT GGAGAGGGTG AAGGTGATGC AACATACGGA 120 AAACTTACCC TTAAATTTATTTGCACTACT GGAAAACTAC 160 CTGTTCCATG GCCAACACTT GTCACTACTT TCTCTTATGG200 TGTTCAATGC TTTTCAAGAT ACCCAGATCA TATGAAACGG 240 CATGACTTTTTCAAGAGTGC CATGCCCGAA GGTTATGTAC 280 AGGAAAGAAC TATATTTTTC AAAGATGACGGGAACTACAA 320 GACACGTGCT GAAGTCAAGT TTGAAGGTGA TACCCTTGTT 360AATAGAATTG AGTTAAAAGG TATTGATTTT AAAGAAGATG 400 GAAACATTCT TGGACACAAATTGGAGTACA ACTATAACCC 440 TCACTATGTG TACATCATGG CAGACAAACA AAAGAATGGT480 ACCAAAGTTA ACTTTCAAGT TCACCACAAC ATTGAAGATG 520 GAAGCGTTCAACTAGCAGAC CATTATCAAC AAAATACTCC 560 AATTGGCGAT GGCCCTGTCC TTTTACCAGACAACCATTAC 600 CTGCACACAC AATCTGCCCT TTCGAAAGAT CCCAACGAAA 640AGAGAGACCA CATGGTCCTT CTTGAGTTTG TAACAGCTGC 680 TGGGATTACA CATGGCATGGATGAACTATA CAAATAA 717 717 NUCLEIC ACID UNKNOWN UNKNOWN 2G3 14ATGAGTAAAG GAGAAGAACT TTTCACTGGA GTTGTCCCAA 40 TTCTTGTTGA ATTAGATGGTGATGTTAATG GGCACAAATT 80 TTCTGTCAGT GGAGAGGGTG AAGGTGATGC AACATACGGA 120AAACTTACCC TTAAATTTAT TTGCACTACT GGAAAACTAC 160 CTGTTCCATG GCCAACACTTGTCACTACTT TCTCTTATGG 200 TGTTCAATGC TTTTCAAGAT ACCCAGATCA TATGAAACGG240 CATGACTTTT TCAAGAGTGC CATGCCCGAA GGTTATGTAC 280 AGGAAAGAACTATATTTTTC AAAGATGACG GGAACTACAA 320 GACACGTGCT GAAGTCAAGT TTGAAGGTGATACCCTTGTT 360 AATAGAATCG AGTTAAAAGG TATTGATTTT AAAGAAGATG 400GAAACATTCT TGGACACAAA TTGGAATACA ACTATAACGA 440 GCACTTGGTG TACATCATGGCAGACAAACA AAAGAATGGT 480 ACCAAAGCTA ACTTTAAAAT TCACCACAAC ATTGAAGATG520 GAGGCGTTCA ACTAGCAGAC CATTATCAAC AAAATACTCC 560 AATTGGCGATGGCCCTGTCC TTTTACCAGA CAACCATTAC 600 CTGCACACAC AATCTGCCCT TTCGAAAGATCCCAACGAAA 640 AGAGAGACCA CATGGTCCTT CTTGAGTTTG TAACAGCTGC 680TGGGATTACA CATGGCATGG ATGAACTATA CAAATAA 717 717 NUCLEIC ACID UNKNOWNUNKNOWN S202H 15 ATGAGTAAAG GAGAAGAACT TTTCACTGGA GTTGTCCCAA 40TTCTTGTTGA ATTAGATGGT GATGTTAATG GGCACAAATT 80 TTCTGTCAGT GGAGAGGGTGAAGGTGATGC AACATACGGA 120 AAACTTACCC TTAAATTTAT TTGCACTACT GGAAAACTAC160 CTGTTCCATG GCCAACACTT GTCACTACTT TCTCTTATGG 200 TGTTCAATGCTTTTCAAGAT ACCCAGATCA TATGAAACGG 240 CATGACTTTT TCAAGAGTGC CATGCCCGAAGGTTATGTAC 280 AGGAAAGAAC TATATTTTTC AAAGATGACG GGAACTACAA 320GACACGTGCT GAAGTCAAGT TTGAAGGTGA TACCCTTGTT 360 AATAGAATCG AGTTAAAAGGTATTGATTTT AAAGAAGATG 400 GAAACATTCT TGGACACAAA TTGGAATACA ACTATAACTC440 ACACAATGTA TACATCATGG CAGACAAACA AAAGAATGGA 480 ATCAAAGTTAACTTCAAAAT TAGACACAAC ATTGAAGATG 520 GAAGCGTTCA ACTAGCAGAC CATTATCAACAAAATACTCC 560 AATTGGCGAT GGCCCTGTCC TTTTACCAGA CAACCATTAC 600CTGCACACAC AATCTGCCCT TTCGAAAGAT CCCAACGAAA 640 AGAGAGACCA CATGGTCCTTCTTGAGTTTG TAACAGCTGC 680 TGGGATTACA CATGGCATGG ATGAACTATA CAAATAA 717717 NUCLEIC ACID UNKNOWN UNKNOWN 14D9 16 ATGAGTAAAG GAGAAGAACTTTTCACTGGA GTTGTCCCAA 40 TTCTTGTTGA ATTAGATGGT GATGTTAATG GGCACAAATT 80TTCTGTCAGT GGAGAGGGTG AAGGTGATGC AACATACGGA 120 AAACTTACCC TTAAATTTATTTGCACTACT GGAAAACTAC 160 CTGTTCCATG GCCAACACTT GTCACTACTT TCTCTTATGG200 TGTTCAATGC TTTTCAAGAT ACCCAGATCA TATGAAACGG 240 CATGACTTTTTCAAGAGTGC CATGCCCGAA GGTTATGTAC 280 AGGAAAGAAC TATATTTTTC AAAGATGACGGGAACTACAA 320 GACACGTGCT GAAGTCAAGT TTGAAGGTGA TACCCTTGTT 360AATAGAATTG AGTTAAAAGG TATTGATTTT AAAGAAGATG 400 GAAACATTCT TGGACACAAATTGGAGTACA ACTATAACCC 440 TCACTGGGTG TACATCATGG CAGACAAACA AAAGAATGGT480 ACCAAAGTTA ACTTTCAAGT TCACCACAAC ATTGAAGATG 520 GAAGCGTTCAACTAGCAGAC CATTATCAAC AAAATACTCC 560 AATTGGCGAT GGCCCTGTCC TTTTACCAGACAACCATTAC 600 CTGCACACAC AATCTGCCCT TTCGAAAGAT CCCAACGAAA 640AGAGAGACCA CATGGTCCTT CTTGAGTTTG TAACAGCTGC 680 TGGGATTACA CATGGCATGGATGAACTATA CAAATAA 717 717 NUCLEIC ACID UNKNOWN UNKNOWN 8H8 17ATGAGTAAAG GAGAAGAACT TTTCACTGGA GTTGTCCCAA 40 TTCTTGTTGA ATTAGATGGTGATGTTAATG GGCACAAATT 80 TTCTGTCAGT GGAGAGGGTG AAGGTGATGC AACATACGGA 120AAACTTACCC TTAAATTTAT TTGCACTACT GGAAAACTAC 160 CTGTTCCATG GCCAACACTTGTCACTACTT TCTCTTATGG 200 TGTTCAATGC TTTTCAAGAT ACCCAGATCA TATGAAACGG240 CATGACTTTT TCAAGAGTGC CATGCCCGAA GGTTATGTAC 280 AGGAAAGAACTATATTTTTC AAAGATGACG GGAACTACAA 320 GACACGTGCT GAAGTCAAGT TTGAAGGTGATACCCTTGTT 360 AATAGAATCG AGTTAAAAGG TATTGATTTT AAAGAAGATG 400GAAACATTCT TGGACACAAA TTGGAATACA ACTATAACCC 440 TCACTGGGTG TACATCATGGCAGACAAACA AAAGAATGGA 480 ATCAAAGTTA ACTTCAAAAT TAGACACAAC ATTGAAGATG520 GAAGCGTTCA ACTAGCAGAC CATTATCAAC AAAATACTCC 560 AATTGGCGATGGCCCTGTCC TTTTACCAGA CAACCATTAC 600 CTGCACACAC AATCTGCCCT TTCGAAAGATCCCAACGAAA 640 AGAGAGACCA CATGGTCCTT CTTGAGTTTG TAACAGCTGC 680TGGGATTACA CATGGCATGG ATGAACTATA CAAATAA 717 1668 NUCLEIC ACID UNKNOWNUNKNOWN CYPRIDINA LUCIFERASE 18 ATGAAGATAA TAATTCTGTC TGTTATATTGGCCTACTGTG 40 TCACCGTCAA CTGTCAAGAT GCATGTCCTG TAGAAGCGGA 80 ACCGCCATCAAGTACACCAA CAGTTCCAAC TTCTTGTGAA 120 GCTAAAGAAG GAGAATGTAT AGATACCAGATGCGCAACAT 160 GTAAACGAGA TATACTATCA GACGGACTGT GTGAAAATAA 200ACCAGGGAAG ACATGCTGTA GAATGTGCCA GTATGTGATT 240 GAATGCAGAG TAGAAGCAGCTGGTTATTTT AGAACGTTTT 280 ACGGCAAAAG ATTTAATTTT CAGGAACCTG GTAAATATGT320 GCTGGCTAGG GGAACCAAGG GTGGCGATTG GTCTGTAACC 360 CTCACCATGGAGAACCTAGA TGGACAGAAG GGAGCTGTGC 400 TGACTAAGAC AACACTGGAG GTTGCAGGAGACGTAATAGA 440 CATTACTCAA GCTACTGCAG ATCCTATCAC AGTTAACGGA 480GGAGCTGACC CAGTTATCGC TAACCCGTTC ACAATTGGTG 520 AGGTGACCAT TGCTGTTGTTGAAATACCGG GCTTCAATAT 560 CACAGTCATC GAATTCTTTA AACTAATCGT GATTGATATT600 CTGGGAGGAA GATCTGTGAG AATTGCTCCA GACACAGCAA 640 ACAAAGGACTGATATCTGGT ATCTGTGGTA ATCTGGAGAT 680 GAATGACGCT GATGACTTTA CTACAGACGCAGATCAGCTG 720 GCGATCCAAC CCAACATAAA CAAAGAGTTC GACGGCTGCC 760CATTCTATGG GAATCCTTCT GATATCGAAT ACTGCAAAGG 800 TCTCATGGAG CCATACAGAGCTGTATGTCG TAACAATATC 840 AACTTCTACT ATTACACTCT ATCCTGCGCC TTCGCTTACT880 GTATGGGAGG AGAAGAAAGA GCTAAACACG TCCTTTTCGA 920 CTATGTTGAGACATGCGCTG CACCGGAAAC GAGAGGAACG 960 TGTGTTTTAT CAGGACATAC TTTCTATGACACATTCGACA 1000 AAGCCAGATA TCAATTCCAG GGCCCATGCA AAGAGCTTCT 1040GATGGCCGCA GACTGTTACT GGAACACATG GGATGTAAAG 1080 GTTTCACATA GAGATGTTGAGTCATACACT GAGGTAGAGA 1120 AAGTAACAAT CAGGAAACAG TCAACTGTAG TAGATCTGAT1160 TGTGGATGGC AAGCAGGTCA AGGTTGGAGG AGTGGATGTA 1200 TCTATCCCGTACAGCTCTGA GAACACATCC ATATACTGGC 1240 AGGATGGAGA CATCCTGACG ACGGCCATCCTACCTGAAGC 1280 TCTCGTCGTT AAGTTCAACT TTAAGCAGCT CCTTGTAGTT 1320CATATCAGAG ATCCATTCGA TGGAAAGACA TGCGGCATAT 1360 GTGGTAACTA TAATCAAGATTCAACTGATG ATTTCTTTGA 1400 CGCAGAAGGA GCATGCGCTC TGACCCCCAA TCCCCCAGGA1440 TGTACAGAGG AGCAGAAACC AGAAGCTGAG CGACTCTGCA 1480 ATAGTCTATTTGATAGTTCT ATCGACGAGA AATGTAATGT 1520 CTGCTACAAG CCGGACCGTA TTGCCCGATGTATGTACGAG 1560 TATTGCCTGA GGGGACAGCA AGGATTCTGT GACCATGCTT 1600GGGAGTTCAA GAAAGAATGC TACATAAAGC ATGGAGACAC 1640 TCTAGAAGTA CCACCTGAATGTCAATAA 1668 717 NUCLEIC ACID UNKNOWN UNKNOWN C6 19 ATGATTAAAGGAGAAGAACT TTTCACTGGA GTTGTCCCAA 40 TTCTTGTTGA ATTAGATGGT GATGTTAATGGGCACAAATT 80 TTCTGTCAGT GGAGAGGGTG AAGGTGATGC AACATACGGA 120 AAACTTACCCTTAAATTTAT TTGCACTACT GGAAAACTAC 160 CTGTTCCATG GCCAACACTT GTCACTACTTTCTCTTATGG 200 TGTTCAATGC TTTTCAAGAT ACCCAGATCA TATGAAACGG 240CATGACTTTT TCAAGAGTGC CATGCCCGAA GGTTATGTAC 280 AGGAAAGAAC TATATTTTTCAAAGATGACG GGAACTACAA 320 GACACGTGCT GAAGTCAAGT TTGAAGGTGA TACCCTTGTT360 AATAGAATCG AGTTAAAAGG TATTGATTTT AAAGATGATG 400 GAAACATTCTTGGACACAAA TTGGAATACA ACTATAACGA 440 GCACTTGGTG TACATCATGG CAGACAAACAAAAGAATGGT 480 ACCAAAGCTA TCTTTCAAGT TCACCACAAC ATTGAAGATG 520GAGGCGTTCA ACTAGCAGAC CATTATCAAC AAAATACTCC 560 AATTGGCGAT GGCCCTGTCCTTTTACCAGA CAACCATTAC 600 CTGCACACAC AATCTGCCCT TTCGAAAGAT CCCAACGAAA640 AGAGAGACCA CATGGTCTTT CTTGAGTTTG TAACAGCTGC 680 TGGGATTACACATGGCATGG ATGAAGTNTA CAAATAA 717 238 AMINO ACID UNKNOWN UNKNOWN 20 MetIle Lys Gly Glu Glu Leu Phe Thr Gly Val Val 1 5 10 Pro Ile Leu Val GluLeu Asp Gly Asp Val Asn Gly 15 20 His Lys Phe Ser Val Ser Gly Glu GlyGlu Gly Asp 25 30 35 Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 4045 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 50 55 60 Val Thr ThrPhe Ser Tyr Gly Val Gln Cys Phe Ser 65 70 Arg Tyr Pro Asp His Met LysArg His Asp Phe Phe 75 80 Lys Ser Ala Met Pro Glu Gly Tyr Val Gln GluArg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 100 105 ArgAla Glu Val Lys Phe Glu Gly Asp Thr Leu Val 110 115 120 Asn Arg Ile GluLeu Lys Gly Ile Asp Phe Lys Asp 125 130 Asp Gly Asn Ile Leu Gly His LysLeu Glu Tyr Asn 135 140 Tyr Asn Glu His Leu Val Tyr Ile Met Ala Asp Lys145 150 155 Gln Lys Asn Gly Thr Lys Ala Ile Phe Gln Val His 160 165 HisAsn Ile Glu Asp Gly Gly Val Gln Leu Ala Asp 170 175 180 His Tyr Gln GlnAsn Thr Pro Ile Gly Asp Gly Pro 185 190 Val Leu Leu Pro Asp Asn His TyrLeu His Thr Gln 195 200 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp205 210 215 His Met Val Phe Leu Glu Phe Val Thr Ala Ala Gly 220 225 IleThr His Gly Met Asp Glu Val Tyr Lys 230 235 717 NUCLEIC ACID UNKNOWNUNKNOWN 21 ATGAGTAAAG GAGAAGAACT TTTCACTGGA GTTGTCCCAA 40 TTCTTGTTGAATTAGATGGT GATGTTAATG GGCACAAATT 80 TTCTGTCAGT GGAGAGGGTG AAGGTGATGCAACATACGGA 120 AAACTTACCC TTAAATTTAT TTGCACTACT GGAAAACTAC 160CTGTTCCATG GCCAACACTT GTCACTACTT TCTCTTATGG 200 TGTTCAATGC TTTTCAAGATACCCAGATCA TATGAAACGG 240 CATGACTTTT TCAAGAGTGC CATGCCCGAA GGTTATGTAC280 AGGAAAGAAC TATATTTTTC AAAGATGACG GGAACTACAA 320 GACACGTGCTGAAGTCAAGT TTGAAGGTGA TACCCTTGTT 360 AATAGAATCG AGTTAAAAGG TATTGATTTTAAAGAAGATG 400 GAAACATTCT TGGACACAAA TTGGAATACA ACTATAACGA 440TCACCAGGTG TACATCATGG CAGACAAACA AAAGAATGGA 480 ATCAAAGCTA ACTTCAAAATTAGACACAAC ATTGAAGATG 520 GAGGCGTTCA ACTAGCAGAC CATTATCAAC AAAATACTCC560 AATTGGCGAT GGGCCCGTCC TTTTACCAGA CAACCATTAC 600 CTGTTTACAACTTCTACTCT TTCGAAAGAT CCCAACGAAA 640 AGAGAGACCA CATGGTCCTT CTTGAGTTTGTAACAGCTGC 680 TGGGATTACA CATGGCATGG ATGAACTATA CAAATAA 717 238 AMINOACID UNKNOWN UNKNOWN 22 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val1 5 10 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly 15 20 His Lys PheSer Val Ser Gly Glu Gly Glu Gly Asp 25 30 35 Ala Thr Tyr Gly Lys Leu ThrLeu Lys Phe Ile Cys 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro ThrLeu 50 55 60 Val Thr Thr Phe Ser Tyr Gly Val Gln Cys Phe Ser 65 70 ArgTyr Pro Asp His Met Lys Arg His Asp Phe Phe 75 80 Lys Ser Ala Met ProGlu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly AsnTyr Lys Thr 100 105 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val 110115 120 Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu 125 130 Asp GlyAsn Ile Leu Gly His Lys Leu Glu Tyr Asn 135 140 Tyr Asn Asp His Gln ValTyr Ile Met Ala Asp Lys 145 150 155 Gln Lys Asn Gly Ile Lys Ala Asn PheLys Ile Arg 160 165 His Asn Ile Glu Asp Gly Gly Val Gln Leu Ala Asp 170175 180 His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 185 190 Val LeuLeu Pro Asp Asn His Tyr Leu Phe Thr Thr 195 200 Ser Thr Leu Ser Lys AspPro Asn Glu Lys Arg Asp 205 210 215 His Met Val Leu Leu Glu Phe Val ThrAla Ala Gly 220 225 Ile Thr His Gly Met Asp Glu Leu Tyr Lys 230 235 238AMINO ACID UNKNOWN UNKNOWN 23 Met Ser Lys Gly Glu Glu Leu Phe Thr GlyVal Val 1 5 10 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly 15 20 HisLys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp 25 30 35 Ala Thr Tyr Gly LysLeu Thr Leu Lys Phe Ile Cys 40 45 Thr Thr Gly Lys Leu Pro Val Pro TrpPro Thr Leu 50 55 60 Val Thr Thr Phe Ser Tyr Gly Val Gln Cys Phe Ser 6570 Arg Tyr Pro Asp His Met Lys Arg His Asp Phe Phe 75 80 Lys Ser Ala MetPro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp GlyAsn Tyr Lys Thr 100 105 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val110 115 120 Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu 125 130 AspGly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 135 140 Tyr Asn Asp His GlnVal Tyr Ile Met Ala Asp Lys 145 150 155 Gln Lys Asn Gly Ile Lys Ala AsnPhe Lys Ile Arg 160 165 His Asn Ile Glu Asp Gly Gly Val Gln Leu Ala Asp170 175 180 His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 185 190 ValLeu Leu Pro Asp Asn His Tyr Leu His Thr Gln 195 200 Ser Ala Leu Ser LysAsp Pro Asn Glu Lys Arg Asp 205 210 215 His Met Val Leu Leu Glu Phe ValThr Ala Ala Gly 220 225 Ile Thr His Gly Met Asp Glu Leu Tyr Lys 230 235238 AMINO ACID UNKNOWN UNKNOWN 24 Met Ser Lys Gly Glu Glu Leu Phe ThrGly Val Val 1 5 10 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly 15 20His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp 25 30 35 Ala Thr Tyr GlyLys Leu Thr Leu Lys Phe Ile Cys 40 45 Thr Thr Gly Lys Leu Pro Val ProTrp Pro Thr Leu 50 55 60 Val Thr Thr Phe Ser Tyr Gly Val Gln Cys Phe Ser65 70 Arg Tyr Pro Asp His Met Lys Arg His Asp Phe Phe 75 80 Lys Ser AlaMet Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp AspGly Asn Tyr Lys Thr 100 105 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr LeuVal 110 115 120 Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu 125 130Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 135 140 Tyr Asn Asp HisAsp Val Tyr Ile Met Ala Asp Lys 145 150 155 Gln Lys Asn Gly Thr Lys AlaAsn Phe Gln Val Arg 160 165 His Asn Ile Glu Asp Gly Gly Val Gln Leu AlaAsp 170 175 180 His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 185 190Val Leu Leu Pro Asp Asn His Tyr Leu His Thr Gln 195 200 Ser Ala Leu SerLys Asp Pro Asn Glu Lys Arg Asp 205 210 215 His Met Val Leu Leu Glu PheVal Thr Ala Ala Gly 220 225 Ile Thr His Gly Met Asp Glu Leu Tyr Lys 230235 238 AMINO ACID UNKNOWN UNKNOWN 25 Met Ser Lys Gly Glu Glu Leu PheThr Gly Val Val 1 5 10 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly15 20 His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp 25 30 35 Ala ThrTyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 40 45 Thr Thr Gly Lys Leu ProVal Pro Trp Pro Thr Leu 50 55 60 Val Thr Thr Phe Ser Tyr Gly Val Gln CysPhe Ser 65 70 Arg Tyr Pro Asp His Met Lys Arg His Asp Phe Phe 75 80 LysSer Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe LysAsp Asp Gly Asn Tyr Lys Thr 100 105 Arg Ala Glu Val Lys Phe Glu Gly AspThr Leu Val 110 115 120 Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu125 130 Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 135 140 Tyr AsnAsp His Asn Val Tyr Ile Met Ala Asp Lys 145 150 155 Gln Lys Asn Gly IleLys Val Asn Phe Lys Ile Arg 160 165 His Asn Ile Glu Asp Gly Ser Val GlnLeu Ala Asp 170 175 180 His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro185 190 Val Leu Leu Pro Asp Asn His Tyr Leu His Thr Gln 195 200 Ser AlaLeu Ser Lys Asp Pro Asn Glu Lys Arg Asp 205 210 215 His Met Val Leu LeuGlu Phe Val Thr Ala Ala Gly 220 225 Ile Thr His Gly Met Asp Glu Leu TyrLys 230 235 238 AMINO ACID UNKNOWN UNKNOWN 26 Met Ser Lys Gly Glu GluLeu Phe Thr Gly Val Val 1 5 10 Pro Ile Leu Val Glu Leu Asp Gly Asp ValAsn Gly 15 20 His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp 25 30 35Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 40 45 Thr Thr Gly LysLeu Pro Val Pro Trp Pro Thr Leu 50 55 60 Val Thr Thr Phe Ser Tyr Gly ValGln Cys Phe Ser 65 70 Arg Tyr Pro Asp His Met Lys Arg His Asp Phe Phe 7580 Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile PhePhe Lys Asp Asp Gly Asn Tyr Lys Thr 100 105 Arg Ala Glu Val Lys Phe GluGly Asp Thr Leu Val 110 115 120 Asn Arg Ile Glu Leu Lys Gly Ile Asp PheLys Glu 125 130 Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 135 140Tyr Asn Asp His His Val Tyr Ile Met Ala Asp Lys 145 150 155 Gln Lys AsnGly Ile Lys Val Asn Phe Lys Ile Arg 160 165 His Asn Ile Glu Asp Gly SerVal Gln Leu Ala Asp 170 175 180 His Tyr Gln Gln Asn Thr Pro Ile Gly AspGly Pro 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu His Thr Gln 195 200Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp 205 210 215 His Met ValLeu Leu Glu Phe Val Thr Ala Ala Gly 220 225 Ile Thr His Gly Met Asp GluLeu Tyr Lys 230 235 238 AMINO ACID UNKNOWN UNKNOWN 27 Met Ser Lys GlyGlu Glu Leu Phe Thr Gly Val Val 1 5 10 Pro Ile Leu Val Glu Leu Asp GlyAsp Val Asn Gly 15 20 His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp 2530 35 Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 40 45 Thr Thr GlyLys Leu Pro Val Pro Trp Pro Thr Leu 50 55 60 Val Thr Thr Phe Ser Tyr GlyVal Gln Cys Phe Ser 65 70 Arg Tyr Pro Asp His Met Lys Arg His Asp PhePhe 75 80 Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 ThrIle Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 100 105 Arg Ala Glu Val LysPhe Glu Gly Asp Thr Leu Val 110 115 120 Asn Arg Ile Glu Leu Lys Gly IleAsp Phe Lys Glu 125 130 Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn135 140 Tyr Asn Asp His Val Val Tyr Ile Met Ala Asp Lys 145 150 155 GlnLys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg 160 165 His Asn Ile Glu AspGly Ser Val Gln Leu Ala Asp 170 175 180 His Tyr Gln Gln Asn Thr Pro IleGly Asp Gly Pro 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu His Thr Gln195 200 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp 205 210 215 HisMet Val Leu Leu Glu Phe Val Thr Ala Ala Gly 220 225 Ile Thr His Gly MetAsp Glu Leu Tyr Lys 230 235 238 AMINO ACID UNKNOWN UNKNOWN 28 Met SerLys Gly Glu Glu Leu Phe Thr Gly Val Val 1 5 10 Pro Ile Leu Val Glu LeuAsp Gly Asp Val Asn Gly 15 20 His Lys Phe Ser Val Ser Gly Glu Gly GluGly Asp 25 30 35 Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 40 45Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 50 55 60 Val Thr Thr PheSer Tyr Gly Val Gln Cys Phe Ser 65 70 Arg Tyr Pro Asp His Met Lys ArgHis Asp Phe Phe 75 80 Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 8590 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 100 105 Arg AlaGlu Val Lys Phe Glu Gly Asp Thr Leu Val 110 115 120 Asn Arg Ile Glu LeuLys Gly Ile Asp Phe Lys Glu 125 130 Asp Gly Asn Ile Leu Gly His Lys LeuGlu Tyr Asn 135 140 Tyr Asn Asp His Gln Val Tyr Ile Met Ala Asp Lys 145150 155 Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg 160 165 His AsnIle Glu Asp Gly Gly Val Gln Leu Ala Asp 170 175 180 His Tyr Gln Gln AsnThr Pro Ile Gly Asp Gly Pro 185 190 Val Leu Leu Pro Asp Asn His Tyr LeuHis Thr Gln 195 200 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp 205210 215 His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly 220 225 Ile ThrHis Gly Met Asp Glu Leu Tyr Lys 230 235 238 AMINO ACID UNKNOWN UNKNOWN29 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val 1 5 10 Pro Ile LeuVal Glu Leu Asp Gly Asp Val Asn Gly 15 20 His Lys Phe Ser Val Ser GlyGlu Gly Glu Gly Asp 25 30 35 Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe IleCys 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 50 55 60 ValThr Thr Phe Ser Tyr Gly Val Gln Cys Phe Ser 65 70 Arg Tyr Pro Asp HisMet Lys Arg His Asp Phe Phe 75 80 Lys Ser Ala Met Pro Glu Gly Tyr ValGln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 100105 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val 110 115 120 Asn ArgIle Glu Leu Lys Gly Ile Asp Phe Lys Glu 125 130 Asp Gly Asn Ile Leu GlyHis Lys Leu Glu Tyr Asn 135 140 Tyr Asn Asp His Thr Val Tyr Ile Met AlaAsp Lys 145 150 155 Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg 160165 His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp 170 175 180 His TyrGln Gln Asn Thr Pro Ile Gly Asp Gly Pro 185 190 Val Leu Leu Pro Asp AsnHis Tyr Leu His Thr Gln 195 200 Ser Ala Leu Ser Lys Asp Pro Asn Glu LysArg Asp 205 210 215 His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly 220225 Ile Thr His Gly Met Asp Glu Leu Tyr Lys 230 235 238 AMINO ACIDUNKNOWN UNKNOWN 30 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val 1 510 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly 15 20 His Lys Phe SerVal Ser Gly Glu Gly Glu Gly Asp 25 30 35 Ala Thr Tyr Gly Lys Leu Thr LeuLys Phe Ile Cys 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 5055 60 Val Thr Thr Phe Ser Tyr Gly Val Gln Cys Phe Ser 65 70 Arg Tyr ProAsp His Met Lys Arg His Asp Phe Phe 75 80 Lys Ser Ala Met Pro Glu GlyTyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr LysThr 100 105 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val 110 115 120Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu 125 130 Asp Gly Asn IleLeu Gly His Lys Leu Glu Tyr Asn 135 140 Tyr Asn Asp His Leu Val Tyr IleMet Ala Asp Lys 145 150 155 Gln Lys Asn Gly Thr Lys Val Asn Phe Gln ValHis 160 165 His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp 170 175 180His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 185 190 Val Leu Leu ProAsp Asn His Tyr Leu His Thr Gln 195 200 Ser Ala Leu Ser Lys Asp Pro AsnGlu Lys Arg Asp 205 210 215 His Met Val Leu Leu Glu Phe Val Thr Ala AlaGly 220 225 Ile Thr His Gly Met Asp Glu Leu Tyr Lys 230 235 238 AMINOACID UNKNOWN UNKNOWN 31 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val1 5 10 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly 15 20 His Lys PheSer Val Ser Gly Glu Gly Glu Gly Asp 25 30 35 Ala Thr Tyr Gly Lys Leu ThrLeu Lys Phe Ile Cys 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro ThrLeu 50 55 60 Val Thr Thr Phe Ser Tyr Gly Val Gln Cys Phe Ser 65 70 ArgTyr Pro Asp His Met Lys Arg His Asp Phe Phe 75 80 Lys Ser Ala Met ProGlu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly AsnTyr Lys Thr 100 105 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val 110115 120 Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu 125 130 Asp GlyAsn Ile Leu Gly His Lys Leu Glu Tyr Asn 135 140 Tyr Asn Asp His Asp ValTyr Ile Met Ala Asp Lys 145 150 155 Gln Lys Asn Gly Thr Lys Val Asn PheGln Val Arg 160 165 His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp 170175 180 His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 185 190 Val LeuLeu Pro Asp Asn His Tyr Leu His Thr Gln 195 200 Ser Ala Leu Ser Lys AspPro Asn Glu Lys Arg Asp 205 210 215 His Met Val Leu Leu Glu Phe Val ThrAla Ala Gly 220 225 Ile Thr His Gly Met Asp Glu Leu Tyr Lys 230 235 238AMINO ACID UNKNOWN UNKNOWN 32 Met Ser Lys Gly Glu Glu Leu Phe Thr GlyVal Val 1 5 10 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly 15 20 HisLys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp 25 30 35 Ala Thr Tyr Gly LysLeu Thr Leu Lys Phe Ile Cys 40 45 Thr Thr Gly Lys Leu Pro Val Pro TrpPro Thr Leu 50 55 60 Val Thr Thr Phe Ser Tyr Gly Val Gln Cys Phe Ser 6570 Arg Tyr Pro Asp His Met Lys Arg His Asp Phe Phe 75 80 Lys Ser Ala MetPro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp GlyAsn Tyr Lys Thr 100 105 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val110 115 120 Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu 125 130 AspGly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 135 140 Tyr Asn Asp His LeuVal Tyr Ile Met Ala Asp Lys 145 150 155 Gln Lys Asn Gly Thr Lys Val AsnPhe Gln Val Arg 160 165 His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp170 175 180 His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 185 190 ValLeu Leu Pro Asp Asn His Tyr Leu His Thr Gln 195 200 Ser Ala Leu Ser LysAsp Pro Asn Glu Lys Arg Asp 205 210 215 His Met Val Leu Leu Glu Phe ValThr Ala Ala Gly 220 225 Ile Thr His Gly Met Asp Glu Leu Tyr Lys 230 235238 AMINO ACID UNKNOWN UNKNOWN 33 Met Ser Lys Gly Glu Glu Leu Phe ThrGly Val Val 1 5 10 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly 15 20His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp 25 30 35 Ala Thr Tyr GlyLys Leu Thr Leu Lys Phe Ile Cys 40 45 Thr Thr Gly Lys Leu Pro Val ProTrp Pro Thr Leu 50 55 60 Val Thr Thr Phe Ser Tyr Gly Val Gln Cys Phe Ser65 70 Arg Tyr Pro Asp His Met Lys Arg His Asp Phe Phe 75 80 Lys Ser AlaMet Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp AspGly Asn Tyr Lys Thr 100 105 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr LeuVal 110 115 120 Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu 125 130Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 135 140 Tyr Asn Pro HisTyr Val Tyr Ile Met Ala Asp Lys 145 150 155 Gln Lys Asn Gly Thr Lys ValAsn Phe Gln Val His 160 165 His Asn Ile Glu Asp Gly Ser Val Gln Leu AlaAsp 170 175 180 His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 185 190Val Leu Leu Pro Asp Asn His Tyr Leu His Thr Gln 195 200 Ser Ala Leu SerLys Asp Pro Asn Glu Lys Arg Asp 205 210 215 His Met Val Leu Leu Glu PheVal Thr Ala Ala Gly 220 225 Ile Thr His Gly Met Asp Glu Leu Tyr Lys 230235 238 AMINO ACID UNKNOWN UNKNOWN 34 Met Ser Lys Gly Glu Glu Leu PheThr Gly Val Val 1 5 10 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly15 20 His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp 25 30 35 Ala ThrTyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 40 45 Thr Thr Gly Lys Leu ProVal Pro Trp Pro Thr Leu 50 55 60 Val Thr Thr Phe Ser Tyr Gly Val Gln CysPhe Ser 65 70 Arg Tyr Pro Asp His Met Lys Arg His Asp Phe Phe 75 80 LysSer Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe LysAsp Asp Gly Asn Tyr Lys Thr 100 105 Arg Ala Glu Val Lys Phe Glu Gly AspThr Leu Val 110 115 120 Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu125 130 Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 135 140 Tyr AsnGlu His Leu Val Tyr Ile Met Ala Asp Lys 145 150 155 Gln Lys Asn Gly ThrLys Ala Asn Phe Lys Ile His 160 165 His Asn Ile Glu Asp Gly Gly Val GlnLeu Ala Asp 170 175 180 His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro185 190 Val Leu Leu Pro Asp Asn His Tyr Leu His Thr Gln 195 200 Ser AlaLeu Ser Lys Asp Pro Asn Glu Lys Arg Asp 205 210 215 His Met Val Leu LeuGlu Phe Val Thr Ala Ala Gly 220 225 Ile Thr His Gly Met Asp Glu Leu TyrLys 230 235 238 AMINO ACID UNKNOWN UNKNOWN 35 Met Ser Lys Gly Glu GluLeu Phe Thr Gly Val Val 1 5 10 Pro Ile Leu Val Glu Leu Asp Gly Asp ValAsn Gly 15 20 His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp 25 30 35Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 40 45 Thr Thr Gly LysLeu Pro Val Pro Trp Pro Thr Leu 50 55 60 Val Thr Thr Phe Ser Tyr Gly ValGln Cys Phe Ser 65 70 Arg Tyr Pro Asp His Met Lys Arg His Asp Phe Phe 7580 Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile PhePhe Lys Asp Asp Gly Asn Tyr Lys Thr 100 105 Arg Ala Glu Val Lys Phe GluGly Asp Thr Leu Val 110 115 120 Asn Arg Ile Glu Leu Lys Gly Ile Asp PheLys Glu 125 130 Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 135 140Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys 145 150 155 Gln Lys AsnGly Ile Lys Val Asn Phe Lys Ile Arg 160 165 His Asn Ile Glu Asp Gly SerVal Gln Leu Ala Asp 170 175 180 His Tyr Gln Gln Asn Thr Pro Ile Gly AspGly Pro 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu His Thr Gln 195 200Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp 205 210 215 His Met ValLeu Leu Glu Phe Val Thr Ala Ala Gly 220 225 Ile Thr His Gly Met Asp GluLeu Tyr Lys 230 235 238 AMINO ACID UNKNOWN UNKNOWN 36 Met Ser Lys GlyGlu Glu Leu Phe Thr Gly Val Val 1 5 10 Pro Ile Leu Val Glu Leu Asp GlyAsp Val Asn Gly 15 20 His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp 2530 35 Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 40 45 Thr Thr GlyLys Leu Pro Val Pro Trp Pro Thr Leu 50 55 60 Val Thr Thr Phe Ser Tyr GlyVal Gln Cys Phe Ser 65 70 Arg Tyr Pro Asp His Met Lys Arg His Asp PhePhe 75 80 Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 ThrIle Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 100 105 Arg Ala Glu Val LysPhe Glu Gly Asp Thr Leu Val 110 115 120 Asn Arg Ile Glu Leu Lys Gly IleAsp Phe Lys Glu 125 130 Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn135 140 Tyr Asn Pro His Trp Val Tyr Ile Met Ala Asp Lys 145 150 155 GlnLys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg 160 165 His Asn Ile Glu AspGly Ser Val Gln Leu Ala Asp 170 175 180 His Tyr Gln Gln Asn Thr Pro IleGly Asp Gly Pro 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu His Thr Gln195 200 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp 205 210 215 HisMet Val Leu Leu Glu Phe Val Thr Ala Ala Gly 220 225 Ile Thr His Gly MetAsp Glu Leu Tyr Lys 230 235 238 AMINO ACID UNKNOWN UNKNOWN 37 Met SerLys Gly Glu Glu Leu Phe Thr Gly Val Val 1 5 10 Pro Ile Leu Val Glu LeuAsp Gly Asp Val Asn Gly 15 20 His Lys Phe Ser Val Ser Gly Glu Gly GluGly Asp 25 30 35 Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 40 45Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 50 55 60 Val Thr Thr PheSer Tyr Gly Val Gln Cys Phe Ser 65 70 Arg Tyr Pro Asp His Met Lys ArgHis Asp Phe Phe 75 80 Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 8590 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 100 105 Arg AlaGlu Val Lys Phe Glu Gly Asp Thr Leu Val 110 115 120 Asn Arg Ile Glu LeuLys Gly Ile Asp Phe Lys Glu 125 130 Asp Gly Asn Ile Leu Gly His Lys LeuGlu Tyr Asn 135 140 Tyr Asn Pro His Trp Val Tyr Ile Met Ala Asp Lys 145150 155 Gln Lys Asn Gly Thr Lys Val Asn Phe Gln Val His 160 165 His AsnIle Glu Asp Gly Ser Val Gln Leu Ala Asp 170 175 180 His Tyr Gln Gln AsnThr Pro Ile Gly Asp Gly Pro 185 190 Val Leu Leu Pro Asp Asn His Tyr LeuHis Thr Gln 195 200 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp 205210 215 His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly 220 225 Ile ThrHis Gly Met Asp Glu Leu Tyr Lys 230 235 238 AMINO ACID UNKNOWN UNKNOWNXAA 93 XAA = Any amino acid XAA 95 XAA = Any amino acid XAA 97 XAA = Anyamino acid XAA 147 XAA = Any amino acid XAA 149 XAA = Any amino acid XAA166 XAA = Any amino acid XAA 168 XAA = Any amino acid XAA 202 XAA = Anyamino acid XAA 204 XAA = Any amino acid XAA 206 XAA = Any amino acid XAA221 XAA = Any amino acid XAA 223 XAA = Any amino acid 38 Met Ser Lys GlyGlu Glu Leu Phe Thr Gly Val Val 1 5 10 Pro Ile Leu Val Glu Leu Asp GlyAsp Val Asn Gly 15 20 His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp 2530 35 Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 40 45 Thr Thr GlyLys Leu Pro Val Pro Trp Pro Thr Leu 50 55 60 Val Thr Thr Phe Ser Tyr GlyVal Gln Cys Phe Ser 65 70 Arg Tyr Pro Asp His Met Lys Arg His Asp PhePhe 75 80 Lys Ser Ala Met Pro Glu Gly Tyr Xaa Gln Xaa Arg 85 90 95 XaaIle Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 100 105 Arg Ala Glu Val LysPhe Glu Gly Asp Thr Leu Val 110 115 120 Asn Arg Ile Glu Leu Lys Gly IleAsp Phe Lys Glu 125 130 Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn135 140 Tyr Asn Xaa His Xaa Val Tyr Ile Met Ala Asp Lys 145 150 155 GlnLys Asn Gly Ile Lys Val Asn Phe Xaa Ile Xaa 160 165 His Asn Ile Glu AspGly Ser Val Gln Leu Ala Asp 170 175 180 His Tyr Gln Gln Asn Thr Pro IleGly Asp Gly Pro 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Xaa Thr Xaa195 200 Ser Xaa Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp 205 210 215 HisMet Val Leu Xaa Glu Xaa Val Thr Ala Ala Gly 220 225 Ile Thr His Gly MetAsp Glu Leu Tyr Lys 230 235 238 AMINO ACID UNKNOWN UNKNOWN XAA 147 XAA =Any amino acid XAA 149 XAA = Any amino acid XAA 161 XAA = Any amino acidXAA 163 XAA = Any amino acid XAA 166 XAA = Any amino acid XAA 167 XAA =Any amino acid XAA 168 XAA = Any amino acid XAA 175 XAA = Any amino acidXAA 202 XAA = Any amino acid 39 Met Ser Lys Gly Glu Glu Leu Phe Thr GlyVal Val 1 5 10 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly 15 20 HisLys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp 25 30 35 Ala Thr Tyr Gly LysLeu Thr Leu Lys Phe Ile Cys 40 45 Thr Thr Gly Lys Leu Pro Val Pro TrpPro Thr Leu 50 55 60 Val Thr Thr Phe Ser Tyr Gly Val Gln Cys Phe Ser 6570 Arg Tyr Pro Asp His Met Lys Arg His Asp Phe Phe 75 80 Lys Ser Ala MetPro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp GlyAsn Tyr Lys Thr 100 105 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val110 115 120 Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu 125 130 AspGly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 135 140 Tyr Asn Xaa His XaaVal Tyr Ile Met Ala Asp Lys 145 150 155 Gln Lys Asn Gly Xaa Lys Xaa AsnPhe Xaa Xaa Xaa 160 165 His Asn Ile Glu Asp Gly Xaa Val Gln Leu Ala Asp170 175 180 His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 185 190 ValLeu Leu Pro Asp Asn His Tyr Leu Xaa Thr Gln 195 200 Ser Ala Leu Ser LysAsp Pro Asn Glu Lys Arg Asp 205 210 215 His Met Val Leu Leu Glu Phe ValThr Ala Ala Gly 220 225 Ile Thr His Gly Met Asp Glu Leu Tyr Lys 230 235

We claim:
 1. A pH-sensitive mutant of the GFP of Aequorea Victoria,wherein a change in pH of 1.9 pH units within the range of pH 5.5 to pH10 results in the attenuation or loss of a peak in the excitationspectrum of the mutant.
 2. The pH-sensitive mutant protein of claim 1,wherein said attenuation of a peak in the excitation spectrum is atleast 85%.
 3. The pH-sensitive mutant protein of claim 1, wherein saidattenuation of a peak in the excitation spectrum is at least 90%.
 4. ThepH-sensitive mutant protein of claim 1, wherein said attenuation of apeak in the excitation spectrum is at least 99%.
 5. The pH-sensitivemutant GFP of claim 1, wherein a change in pH of 1.9 pH units within therange of pH 5.5 to pH 10 results in the loss of a peak in the excitationspectrum of the mutant.
 6. A pH-sensitive mutant of the GFP of AequoreaVictoria wherein a change in pH of 1.9 pH units within the range of pH5.5 to pH 10 results in a decrease in fluorescence at a first peak ofthe excitation spectrum and an increase in fluorescence at a second peakof the excitation spectrum.
 7. The pH-sensitive mutant GFP of claim 6,wherein said decrease in fluorescence at a first peak in the excitationspectrum is about 28% or more.
 8. The pH-sensitive mutant GFP of claim6, wherein said decrease in fluorescence at a first peak in theexcitation spectrum is about 37% or more.
 9. The pH-sensitive mutant GFPof claim 6, wherein said decrease in fluorescence at a first peak in theexcitation spectrum is about 46% or more.
 10. The pH-sensitive mutantGFP of any one of claims 6-9, wherein said increase in fluorescence at asecond peak in the excitation spectrum is about 22% or more.
 11. ThepH-sensitive mutant GFP of any one of claims 6-9, wherein said increasein fluorescence at a second peak in the excitation spectrum is about 68%or more.
 12. The pH-sensitive mutant GFP of any one of claims 6-9,wherein said increase in fluorescence at a second peak in the excitationspectrum is about 158% or more.
 13. A pH-sensitive mutant of the GFP ofAequorea Victoria wherein at least one substitution is made at an aminoacid position adjacent to one or more amino acids selected from thegroup consisting of the amino acids at positions 94, 96, 148, 167, 203,205 and
 222. 14. A pH-sensitive GFP according to any one of claims 1, 5,or 6, wherein at least one substitution is made at an amino acidposition selected from the group consisting of positions 147, 149, 161,163, 166, 167, 168, 175 and
 202. 15. A pH-sensitive mutant proteinaccording to claim 1 or claim 5, wherein at least one of the mutationsis selected from the group consisting of Ser147Glu, Ser147Pro,Asn149Val, Asn149Gln, Asn149Thr, Asn149Leu, Asn149Asp, Asn149Tyr,Asn149Trp, Lys166Gln, Ile167Val, Arg168His, and Ser202His.
 16. ApH-sensitive mutant protein according to any one of claims 1, 5, or 6,wherein at least one of the mutations is selected from the groupconsisting of Ser147Asp, Asn149Gln, Asn149Asp, Lys166Gln, Ile167Val andSer202His.
 17. A pH-sensitive mutant protein according to claim 1 orclaim 5, wherein the mutations comprise Ser147Asp and Asn149Gln.
 18. ApH-sensitive mutant protein according to claim 17 wherein the mutationsfurther comprise Val163Ala and Ser175Gly.
 19. A pH-sensitive mutantprotein according to claim 6 wherein the mutations comprise Ser147Asp,Asn149Asp, Lys166Gln, Ile167Val and Ser202His.
 20. A pH-sensitive mutantprotein according to claim 19 wherein the mutations further compriseVal163Ala and Ser175Gly.
 21. A pH-sensitive GFP according to claim 1,wherein an attenuation of an excitation peak at 395 nm and a loss of anexcitation peak at 475 nm occurs upon a decrease of pH.
 22. ApH-sensitive GFP according to claim 21 selected from the groupconsisting of 1D10 (SEQ ID NO:25), 2F10 (SEQ ID NO:26), 2H2 (SEQ IDNO:27), 1B11 (SEQ ID NO:28), 1B11t (SEQ ID NO:23), 8F6 (SEQ ID NO:29),8F3 (SEQ ID NO:22), and 19E10 (SEQ ID NO:30).
 23. A pH-sensitive GFPaccording to claim 6 which, in response to a decrease in pH, exhibitsdecreased fluorescence due to excitation at 395 nm and increasedfluorescence due to excitation at 475 nm.
 24. A pH-sensitive GFPaccording to claim 23 selected from the group consisting of 14E12 (SEQID NO:31), 14E12t (SEQ ID NO:24), 14C9 (SEQ ID NO:32), 14C8 (SEQ NO:33),2G3 (SEQ ID NO:34), S202H (SEQ ID NO:35), 14D9 (SEQ ID NO:37), C6 (SEQID NO:20) and 8H8 (SEQ ID NO:36).
 25. A fusion protein comprising thepH-sensitive GFP according to any one of claims 1 and 5-9, and at leastone other amino acid sequence.
 26. The fusion protein according to claim25 wherein said other amino acid sequence targets the fusion protein toa cell.
 27. The fusion protein of claim 25 wherein said other amino acidsequence targets said fusion protein to synaptic vesicle membranes. 28.The fusion protein of claim 25 wherein said other amino acid sequence isa synaptic vesicle protein.
 29. The fusion protein of claim 28 whereinsaid other amino acid sequence is chosen from the group consisting ofsynaptotagmin and VAMP/synaptobrevin.
 30. A pH-sensitive mutant proteinaccording to claim 22, wherein the protein is 8F3 (SEQ ID NO:22).
 31. ApH-sensitive mutant protein according to claim 24, wherein the proteinis C6 (SEQ ID NO:20).
 32. A fusion protein comprising the pH-sensitivemutant protein according to claim 30 or claim 31, and at least one otheramino acid sequence.